PROCESS FOR PRODUCING v-COELENTERAZINE COMPOUNDS

ABSTRACT

A simple process for producing v-coelenterazine compounds has been desired. Described is a process for producing a v-coelenterazine compound represented by general formula (II) comprising (1) the step of reacting a compound of general formula (VIII) with a methyltriphenylphosphonium salt in the presence of a base to give a compound represented by general formula (IX), (2) the step of performing a ring-closing metathesis reaction on any one selected from the group consisting of the compound represented by general formula (IX) and a compound of general formula (X) which is the compound of general formula (IX) wherein the amino is protected with R 5 , and then deprotecting R 4  and, if any, R 5  to give a v-coelenteramine compound represented by general formula (XIV), and (3) the step of reacting the compound of general formula (XIV) with a compound represented by general formula (XV) to give the compound of general formula (II).

TECHNICAL FIELD

The present invention relates to a process for producingv-coelenterazine compounds, and so on.

BACKGROUND ART

The development of new drugs is still being actively pursued all overthe world, but it is difficult to conclude that new drugs could beefficiently developed as compared to the efforts involved in thedevelopment. This is believed to be due to yet insufficientunderstanding of biomolecular functions. In fact, even the currentlyavailable drugs remain yet unclear, in many cases, about how they actupon the body to exhibit their pharmaceutical effects. For this reason,it is required for the development of new drugs to synthesize a numberof candidate compounds. Accordingly, if the details of biomolecularmechanism involved in diseases can be clarified, the development of newdrugs will be greatly streamlined and such is expected to contribute tothe development of a groundbreaking molecular target drug. Recently,trace analysis has become required for the same subject in animalexperiments with a new drug. It has thus been strongly desired todevelop a noninvasive in vivo imaging technique also from moral andethical aspects of laboratory animals.

In recent years, as represented by green fluorescence protein (GFP), theimaging technology that visualizes molecules involved in life phenomenahas been playing an important role in the medical and biological fieldsas an innovative technique for elucidating biomolecular functions. Onthe other hand, techniques for imaging the desired molecules in livinganimals (in vivo molecular imaging) are still less developed and, aboveall, it has been actively investigated to develop molecular probesavailable for fluorescent or luminescent imaging in the near-infraredregion (wavelength of 700 nm or longer) which is able to permeatethrough the living tissues (K. Kiyose et al., Chem. Asian J 2008, 3,506).

The present inventors thought that if a practical near-infraredbioluminescence system is newly developed, a breakthrough in vivoimaging technology would be provided and have continued studies,focusing anew on coelenterazine (CTZ, 1) which is a typicalbioluminescence substrate from a long time ago.

CTZ is commonly used as a luminescence substrate in many marineorganisms, including photoprotein aequorin form jellyfish, Renillaluciferase from sea pansy, Gaussia luciferase from copepoda, Oplophorusluciferase from decapoda, etc. Accordingly, if a near-infraredluminescent CTZ compound (CTZ and its analogues) available for any ofluciferases can be developed by modifying the structure of CTZ, it isexpected that a luminescence imaging method applicable also to livinganimals could be provided for the detection with high sensitivity of invivo localization of a target protein and its absolute quantity andmetabolic rate, a promoter or enhancer activity of the target protein,life phenomena of a target cell accompanied by in vivo localization,etc.

Approximately 50 kinds of CTZ compounds have been synthesized so far.Some of them were examined for their substrate specificity in severalbioluminescence systems.

Among them, v-coelenterazine (v-CTZ, 2) first reported by Shimomura,Kishi, et al. (O, Shimomura, Y. Kishi, et al., Biochem. J. 1988, 251,405) was later studied for the luminescence properties of aequorin,Renilla luciferase and Oplophorus luciferase by Inouye and Shimomura. Asa result, it was found that when used as a substrate for Renillaluciferase, the maximum emission wavelength was shifted toward thelonger wavelength side, i.e., from 475 nm (blue emission) to 512 nm(green emission) by about 40 nm (S. Inouye & O, Shimomura, Biochem.Biophys. Res. Commun. 1997, 233, 349). It was also revealed that a part(˜5%) of this emission spectrum distribution reached the near-infraredregion (>700 nm).

In addition, A. M. Loenig et al. recently achieved a longer wavelengthshift by modifying the amino acid sequence of Renilla luciferase (A. M.Loenig et at, Nature Methods 2007, 4, 641). More specifically, theyreported that the maximum emission wavelength was shifted to 547 nm(green emission) when v-CTZ was used as a substrate for the modifiedRenilla luciferase. In fact, they also succeeded in imaging in livingmice by utilizing this luminescence system.

In order to establish a practical in vivo imaging system, however, it isdesired to obtain an emission peak at a longer wavelength region. It isfurther pointed out that v-CTZ is easily oxidized and a problem alsoarises in stability (G. Stepanyuk et al., Anal. Bioanal. Chem. 2010,398, 1809). It is therefore an important task to create a novelsubstrate practically applicable to the CTZ near-infraredbioluminescence system.

Furthermore, a process for producing v-CTZ has not been hithertoreported in detail, and it is also desired to establish the process.

DISCLOSURE OF INVENTION

Under the foregoing circumstances, a simple process for producingv-coelenterazine compounds has been desired.

In order to solve the problems described above, the present inventorshave made extensive investigations and as a result, have succeeded indeveloping a simple and flexible process for producing v-coelenterazine,which is applicable also to the production of a variety ofv-coelenterazine analogues.

That is, the present invention provides the process for producing thefollowing v-coelenterazine compounds, and so on.

[1] A process for producing a v-coelenteramine compound represented bygeneral formula (XIV):

(wherein R¹ is hydrogen, a halogen, a substituted or unsubstitutedhydrocarbon group, or a substituted or unsubstituted heterocyclicgroup), which comprises:

(1) the step of reacting a compound represented by general formula(VIII):

(wherein R¹ is the same as defined above and R⁴ is a protecting group)with a methyltriphenylphosphonium salt in the presence of a base to givea compound represented by general formula (IX):

(wherein R¹ and R⁴ are the same as defined above), and,

(2) the step of performing a ring-closing metathesis reaction on any oneselected from the group consisting of the compound represented bygeneral formula (IX) and a compound represented by general formula (X)which is the compound represented by general formula (IX) wherein theamino is protected with R⁵:

(wherein R¹ and R⁴ are the same as defined above and R⁵ is a protectinggroup), and then deprotecting R⁴ and, if any, R⁵.

[2] A process for producing a v-coelenterazine compound represented bygeneral formula (II):

(wherein:

R¹ is hydrogen, a halogen, a substituted or unsubstituted hydrocarbongroup, or a substituted or unsubstituted heterocyclic group,

each of R² and R³ independently represents hydrogen, hydroxy, analkoxyl, a halogen, or a substituted or unsubstituted hydrocarbongroup), which comprises:

(1) the step of reacting a compound represented by general formula(VIII):

(wherein R¹ is the same as defined above and R⁴ is a protecting group)with a methyltriphenylphosphonium salt in the presence of a base to givea compound represented by general formula (IX):

(wherein R¹ and R⁴ are the same as defined above),

(2) the step of performing a ring-closing metathesis reaction on any oneselected from the group consisting of the compound represented bygeneral formula (IX) and a compound represented by general formula (X)which is the compound represented by general formula (IX) wherein theamino is protected with R⁵:

(wherein R¹ and R⁴ are the same as defined above and R⁵ is a protectinggroup), and then deprotecting R⁴ and if any, R⁵ to give av-coelenteramine compound represented by general formula (XIV):

(wherein R¹ is the same as defined above), and,

(3) the step of reacting the compound represented by general formula(XIV) with a compound represented by general formula (XV):

(wherein each of R^(2′) and R^(3′) independently represents hydrogen,hydroxy, an alkoxyl, a halogen, a hydrocarbon group, or a hydroxy groupprotected with a protecting group) to give the compound represented bygeneral formula (II).

[3] The method according to [1] or [2] described above, wherein at leastone base selected from the group consisting of n-butyl lithium,potassium tert-butoxide, sodium methoxide, sodium ethoxide and lithiumdiisopropylamide is used as the base in the step (1) above.

[4] The method according to any one of [1] to [3] described above,wherein at least one solvent selected from the group consisting oftetrahydrofuran, diethyl ether, cyclopropyl methyl ether, tert-butylmethyl ether, dioxane and toluene is used in the step (1) above.

[5] The method according to any one of [1] to [4] above, wherein thereaction temperature and reaction time in the step (1) above are set at0° C. to 40° C. for an hour to 4 hours.

[6] The method according to any one of [1] to [5] above, wherein aHoveyda-Grubbs second generation catalyst is used as the catalyst forthe ring-closing metathesis reaction in the step (2) above.

[7] The method according to any one of [1] to [6] above, wherein atleast one solvent selected from the group consisting of dichloroethane,dichloromethane, chloroform, trichloroethane, tetrachloroethane,benzene, toluene, xylene, monochlorobenzene, dichlorobenzene, hexane,heptane, octane, tetrahydrofuran, dioxane, diethyl ether, dibutyl ether,diisopropyl ether and dimethoxyethane.

[8] The method according to any one of [1] to [7] above, wherein thereaction temperature and reaction time of the ring-closing metathesisreaction in the step (2) above are set at 25° C. to 110° C. for an hourto 48 hours.

[9] The method according to any one of [1] to [8] above, wherein R¹ ishydrogen, a halogen, a substituted or unsubstituted aryl, a substitutedor unsubstituted arylalkyl, a substituted or unsubstituted arylalkenyl,an alkyl which may optionally be substituted with an alicyclic group, analkenyl which may optionally be substituted with an alicyclic group, analicyclic group, a heterocyclic group, or an alkynyl which mayoptionally be substituted with an alicyclic group, in the formulaeabove.

[10] The method according to any one of [1] to [9] above, wherein eachof R² and R³ independently represents hydrogen, hydroxy, a halogen, analkyl having 1 to 4 carbon atoms which may optionally substituted withan alicyclic group, trifluoromethyl or an alkoxyl, in the formula above.

[11] The method according to any one of [1] to [10] above, wherein R⁴ ismethyl, methoxymethyl, tetrahydropyranyl, benzyl, 4-methoxybenzyl,tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl,phenyldimethylsilyl, tert-butyldiphenylsilyl or triisopropylsilyl, inthe formulae above.

[12] The method according to any one of [1] to [11] above, wherein R⁵ isacetyl, benzoyl, p-tosyl, tert-butoxycarbonyl or benzyloxycarbonyl, inthe formulae above.

[13] The method according to any one of [2] to [12] above, wherein eachof the protecting groups for the hydroxy groups for R^(2′) and R^(3′)independently represents tert-butyldimethylsilyl, methoxymethyl,tetrahydropyranyl, trimethylsilyl, phenyldimethylsilyl, triethylsilyl,phenyldimethylsilyl, tert-butyldiphenylsilyl or triisopropylsilyl, inthe formula above.

[14] The method according to any one of [1] to [13] above, wherein thecompound represented by general formula (VIII) above is obtained by:

(1) reacting 2-amino-3,5-dibromo-6-chloropyrazine with R¹MgX (wherein R¹is the same as defined above and X is a halogen) and ZnCl₂ in thepresence of a palladium catalyst to give the compound represented bygeneral formula (V):

(wherein R¹ is the same as defined above),

(2) reacting the compound represented by general formula (V) above witha compound represented by general formula (VI):

(wherein R⁴ is the same as defined above), in the presence of apalladium catalyst and a base to give the compound represented bygeneral formula (VII):

(wherein R¹ and R⁴ are the same as defined above), and,

(3) reacting the compound represented by general formula (VII) withtributyl(vinyl)tin in the presence of a palladium catalyst.

According to the present invention, there is provided a process forproducing the v-coelenterazine compounds in a simple manner. In apreferred embodiment of the present invention, the v-coelenterazinecompounds can be produced in a high yield.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail.

1. v-Coelenterazine Compound

The present invention provides the compounds represented by generalformula (II) below (v-coelenterazine compounds of the presentinvention).

In the formula:

R¹ is hydrogen, a halogen, a substituted or unsubstituted hydrocarbongroup, or a substituted or unsubstituted heterocyclic group,

each of R² and R³ independently represents hydrogen, hydroxy, analkoxyl, a halogen, or a substituted or unsubstituted hydrocarbon group.

In a preferred embodiment of the present invention, the substituted orunsubstituted hydrocarbon group and the substituted or unsubstitutedheterocyclic group in R¹ to R³ do not substantially inhibit any of thereactions in the process for producing the v-coelenterazine compounds orv-coelenteramine compounds of the present invention.

In a further preferred embodiment of the present invention, in theformulae described above,

R¹ is hydrogen, a halogen, a substituted or unsubstituted aryl, asubstituted or unsubstituted arylalkyl, a substituted or unsubstitutedarylalkenyl, an alkyl which may optionally be substituted with analicyclic group, an alkenyl which may optionally be substituted with analicyclic group, an alicyclic group, a heterocyclic group or an alkynylwhich may optionally be substituted with an alicyclic group, and, eachof R² and R³ independently represents hydrogen, hydroxy, a halogen, analkyl having 1 to 4 carbon atoms which may optionally substituted withan alicyclic group, trifluoromethyl or an alkoxyl.

The “halogen” for R¹ includes, for example, fluorine, chlorine, bromineand iodine. In a preferred embodiment of the present invention, the“halogen” is fluorine.

The “substituted or unsubstituted aryl” for R¹ includes, for example, anaryl having 1 to 5 substituents or an unsubstituted aryl. Thesubstituent is at least one selected from the group consisting of ahalogen (fluorine, chlorine, bromine, iodine, etc.), hydroxy, an alkylhaving 1 to 6 carbon atoms, an alkoxyl having 1 to 6 carbon atoms, anamino, a dialkylamino having 1 to 6 carbon atoms, and the like. In someembodiments of the present invention, the substituent is hydroxy.Specific examples of the “substituted or unsubstituted aryl” includephenyl, p-hydroxyphenyl, p-aminophenyl, p-dimethylaminophenyl, etc. Insome embodiments of the present invention, the “substituted orunsubstituted aryl” is an unsubstituted aryl, e.g., phenyl, etc.

The “substituted or unsubstituted arylalkyl” for R¹ includes, forexample, an arylalkyl having 7 to 10 carbon atoms which contains 1 to 5substituents, or an unsubstituted arylalkyl having 7 to 10 carbon atoms.Examples of the substituent are a halogen (fluorine, chlorine, bromine,iodine, etc.), hydroxy, an alkyl having 1 to 6 carbon atoms, an alkoxylhaving 1 to 6 carbon atoms, an amino, a dialkylamino having 1 to 6carbon atoms, and the like. The “substituted or unsubstituted arylalkyl”includes, for example, benzyl, α-hydroxybenzyl, phenylethyl,p-hydroxybenzyl, p-dimethylaminobenzyl, etc., preferably, benzyl,α-hydroxybenzyl, phenylethyl, etc. In some embodiments of the presentinvention, the “substituted or unsubstituted arylalkyl” is benzyl.

The “substituted or unsubstituted arylalkenyl” for R¹ includes, forexample, an arylalkenyl having 8 to 10 carbon atoms which contain 1 to 5substituents, or an unsubstituted arylalkenyl having 8 to 10 carbonatoms. Examples of the substituent are a halogen (fluorine, chlorine,bromine, iodine, etc.), hydroxy, an alkyl having 1 to 6 carbon atoms, analkoxyl having 1 to 6 carbon atoms, an amino, a dialkylamino having 1 to6 carbon atoms, and the like. Examples of the “substituted orunsubstituted arylalkenyl” include phenylvinyl, p-hydroxyphenylvinyl,p-dimethylaminophenylvinyl, etc. In some embodiments of the presentinvention, the “substituted or unsubstituted arylalkenyl” is anunsubstituted arylalkenyl, e.g., phenylvinyl, etc.

The “alkyl which may optionally be substituted with an alicyclic group”for R¹ is, for example, an unsubstituted straight or branched alkylhaving 1 to 4 carbon atoms, or an unsubstituted straight or branchedalkyl having 1 to 4 carbon atoms which is substituted with, e.g., 1 to10 alicyclic groups. Examples of the alicyclic group are cyclohexyl,cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, thealicyclic group is cyclohexyl, cyclopentyl, adamantyl, etc. Examples ofthe “alkyl which may optionally be substituted with an alicyclic group”are methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, cyclobutylmethyl,cyclopropylmethyl, etc., preferably, methyl, ethyl, propyl,2-methylpropyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl,cyclohexylethyl, etc. In some embodiments of the present invention, the“alkyl which may optionally be substituted with an alicyclic group” is astraight alkyl which may optionally be substituted with an alicyclicgroup, and examples include methyl, ethyl, propyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc.

The “alkenyl which may optionally be substituted with an alicyclicgroup” for R¹ is an unsubstituted straight or branched alkenyl having 2to 6 carbon atoms, or a straight or branched alkenyl having 2 to 6carbon atoms which is substituted with, e.g., 1 to 10 alicyclic groups.Examples of the alicyclic group are cyclohexyl, cyclopentyl, adamantyl,cyclobutyl, cyclopropyl, etc. Preferably, the alicyclic group iscyclohexyl, cyclopentyl, adamantyl, etc. Examples of the “alkenyl whichmay optionally be substituted with an alicyclic group” include vinyl,1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,2-methylpropenyl, etc., preferably, 2-methylpropenyl, etc.

Examples of the “alicyclic group” for R¹ include cyclohexyl,cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, thealicyclic group is cyclohexyl, etc.

The “heterocyclic group” for R¹ is, for example, a group formed by a 5-to 7-membered ring containing 1 to 3 atoms selected from the groupconsisting of N, O and S as the atoms for forming the ring, in additionto carbons and bound through carbon, a group formed by condensing atleast two such rings and bound through carbon, or a group formed bycondensing such rings with a benzene ring and bound through carbon.Examples of the “heterocyclic group” are thiophen-2-yl, 2-furanyl,4-pyridyl, etc. In some embodiments of the present invention, the“heterocyclic group” is a heterocyclic group containing sulfur, e.g.,thiophen-2-yl.

The “alkynyl which may optionally be substituted with an alicyclicgroup” for R¹ is an unsubstituted straight or branched alkynyl having 2to 6 carbon atoms, or a substituted straight or branched alkynyl having2 to 6 carbon atoms which is optionally substituted with, e.g., 1 to 10alicyclic groups. Examples of the alicyclic group are cyclohexyl,cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, thealicyclic group is cyclohexyl, cyclopentyl, adamantyl, etc. Examples ofthe “alkynyl which may optionally be substituted with an alicyclicgroup” are ethynyl, propynyl, butynyl, 2-methylpropynyl, etc.,preferably, 2-methylpropynyl, etc.

In a preferred embodiment of the present invention, R¹ is phenyl,p-hydroxyphenyl, benzyl, α-hydroxybenzyl, phenylethyl, phenylvinyl,cyclohexyl, cyclohexylmethyl, cyclohexylethyl, methyl, ethyl, propyl,2-methylpropyl, 2-methylpropenyl, adamantylmethyl, cyclopentylmethyl orthiophen-2-yl. In a more preferred embodiment of the present invention,R¹ is benzyl.

The “halogen” for R² includes, for example, fluorine, chlorine, bromineand iodine. In a preferred embodiment of the present invention, the“halogen” is fluorine.

The “alkyl having 1 to 4 carbon atoms which may optionally besubstituted with an aliphatic group” for R² is, for example, anunsubstituted straight or branched alkyl having 1 to 4 carbon atoms or astraight or branched alkyl having 1 to 4 carbon atoms which issubstituted with, e.g., 1 to 10 alicyclic groups. Examples of thealicyclic group are cyclohexyl, cyclopentyl, adamantyl, cyclobutyl,cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl,cyclopentyl, adamantyl, etc. Examples of the “alkyl which may optionallybe substituted with an alicyclic group” are methyl, ethyl, propyl,2-methylpropyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl,cyclohexylethyl, cyclobutylmethyl, cyclopropylmethyl, etc., preferably,methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc. In someembodiments of the present invention, the “alkyl which may optionally besubstituted with an alicyclic group” is a straight alkyl which mayoptionally be substituted with an alicyclic group, and examples includemethyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, etc.

The “alkoxyl” for R² includes, for example, a straight or branchedalkoxy having 1 to 6 carbon atoms. Examples of the “alkoxy” are methoxy,ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy,sec-butoxy, tert-butoxy, n-pentyloxy, iso-pentyloxy, sec-pentyloxy,1,1-dimethylpropyloxy, 1,2-dimethylpropoxy, 2,2-dimethylpropyloxy,n-hexoxy, 1-ethylpropoxy, 2-ethylpropoxy, 1-methylbutoxy,2-methylbutoxy, iso-hexoxy, 1-methyl-2-ethylpropoxy,1-ethyl-2-methylpropoxy, 1,1,2-trimethylpropoxy, 1,1,2-trimethylpropoxy,1-propylpropoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy,2,2-dimethylbutoxy, 2,3-dimethylbutyloxy, 1,3-dimethylbutyloxy,2-ethylbutoxy, 1,3-dimethylbutoxy, 2-methylpentoxy, 3-methylpentoxy,hexyloxy, etc. In some embodiments of the present invention, the“alkoxy” is methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy,n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, etc., preferably,methoxy.

In a preferred embodiment of the present invention, R² is hydrogen,hydroxy, methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, trifluoromethyl, methoxy, ethoxy,n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy ortert-butoxy. In a more preferred embodiment of the present invention, R²is hydroxy or trifluoromethyl.

The “halogen” for R³ includes, for example, fluorine, chlorine, bromineand iodine. In a preferred embodiment of the present invention, the“halogen” is fluorine.

The “alkyl having 1 to 4 carbon atoms which may optionally besubstituted with an alicyclic group” for R³ includes, for example, anunsubstituted straight or branched alkyl having 1 to 4 carbon atoms or asubstituted straight or branched alkyl having 1 to 4 carbon atoms whichis substituted with, e.g., 1 to 10 alicyclic groups. Examples of thealicyclic group are cyclohexyl, cyclopentyl, adamantyl, cyclobutyl,cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl,cyclopentyl, adamantyl, etc. Examples of the “alkyl which may optionallybe substituted with an alicyclic group” are methyl, ethyl, propyl,2-methylpropyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl,cyclohexylethyl, cyclobutylmethyl, cyclopropylmethyl, etc., preferably,methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc. In someembodiments of the present invention, the “alkyl which may optionally besubstituted with an alicyclic group” is a straight alkyl which mayoptionally be substituted with an alicyclic group, and examples includemethyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, etc.

The “alkoxyl” for R³ includes, for example, a straight or branched“alkoxy” having 1 to 6 carbon atoms and examples of the “alkoxy” aremethoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy,iso-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy, iso-pentyloxy,sec-pentyloxy, 1,1-dimethylpropyloxy, 1,2-dimethylpropoxy,2,2-dimethylpropyloxy, n-hexoxy, 1-ethylpropoxy, 2-ethylpropoxy,1-methylbutoxy, 2-methylbutoxy, iso-hexoxy, 1-methyl-2-ethylpropoxy,1-ethyl-2-methylpropoxy, 1,1,2-trimethylpropoxy, 1,1,2-trimethylpropoxy,1-propylpropoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy,2,2-dimethylbutoxy, 2,3-dimethylbutyloxy, 1,3-dimethylbutyloxy,2-ethylbutoxy, 1,3-dimethylbutoxy, 2-methylpentoxy, 3-methylpentoxy,hexyloxy, etc. In some embodiments of the present invention, the“alkoxy” includes methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy,n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and preferably, methoxy.

In a preferred embodiment of the present invention, R³ is hydrogen,hydroxy, fluorine, methyl, ethyl, propyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, trifluoromethyl,methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy,iso-butoxy, sec-butoxy or tert-butoxy. In a more preferred embodiment ofthe present invention, R³ is hydrogen.

In some embodiments of the present invention, in the general formula(II):

R¹ is benzyl,

R² is hydroxy or trifluoromethyl, and,

R³ is hydrogen.

In a certain embodiment of the present invention, the compoundrepresented by general formula (II) is the compound represented by theformula described below (v-coelenterazine).

In another embodiment of the present invention, the compound representedby general formula (II) is the compound represented by the followingformula (cf3-v-coelenterazine).

The present invention further provides the compounds represented bygeneral formula (XIV) below (the v-coelenteramine compounds of thepresent invention):

(wherein R¹ is the same as described for the general formula (II)above).

In a certain embodiment of the present invention, the compoundrepresented by general formula (XIV) is the compounds represented by thefollowing formula (v-coelenteramine).

2. Process for Producing v-Coelenteramine Compounds or v-CoelenterazineCompounds of the Invention

The process for producing the compound represented by general formula(XIV) (process for producing the v-coelenteramine compounds of thepresent invention) and the process for producing the compoundrepresented by general formula (II) (process for producing thev-coelenterazine compounds of the present invention) are describedbelow.

In the production processes described below, R¹ to R³ are the same asdescribed above.

Each of R^(2′) and R^(3′) independently represents hydrogen, hydroxy, analkoxyl, a halogen, a substituted or unsubstituted hydrocarbon group ora protected hydroxy, etc. The protecting group in the “hydroxy protectedwith a protecting group” for R^(2′) and R^(3′) is, for example,tert-butyldimethylsilyl, methoxymethyl, tetrahydropyranyl,trimethylsilyl, triethylsilyl, phenyldimethylsilyl,tert-butyldiphenylsilyl or triisopropylsilyl, preferably,tert-butyldimethylsilyl.

The “alkoxyl,” “halogen” and “substituted or unsubstituted hydrocarbongroup” for R^(2′) and R^(3′) are the same as described for R² and R³.

Preferably, R^(2′) is tert-butyldimethylsilyloxy. Preferably, R^(3′) ishydrogen.

R⁴ is a protecting group, e.g., methyl, methoxymethyl,tetrahydropyranyl, benzyl, 4-methoxybenzyl, tert-butyldimethylsilyl,trimethylsilyl, triethylsilyl, phenyldimethylsilyl,tert-butyldiphenylsilyl or triisopropylsilyl, and preferably, methyl.

R⁵ is a protecting group, e.g., acetyl, benzoyl, p-tosyl,tert-butoycarbonyl or benzyloxycarbonyl. Preferably, R⁵ is acetyl.

X is a halogen, e.g., fluorine (F⁻), chlorine (Cl⁻), bromine (Br⁻) oriodine (I⁻). Preferably, X is bromine (Br⁻).

2.1. Process for Producing the Compound Represented by General Formula(XIV)

The compound represented by general formula (XIV) can be produced fromthe compound represented by general formula (VIII) as follows.

(1) In the first step, the formyl in the compound represented by generalformula (VIII) is methylated via a Wittig reaction. More specifically,the compound represented by general formula (VIII) is reacted with amethyltriphenylphosphonium salt (Ph₃P⁺CH₃X⁻) in the presence of a baseto give the compound represented by general formula (IX).

The methyltriphenylphosphonium salt is preferablymethyltriphenylphosphonium bromide.

Specific examples of the base used in the Wittig reaction are n-butyllithium (n-BuLi), potassium tert-butoxide, sodium methoxide, sodiumethoxide, lithium diisopropylamide, etc.

Specific examples of the solvents used in the reaction includetetrahydrofuran, diethyl ether, cyclopropyl methyl ether, tert-butylmethyl ether, dioxane, toluene, etc. These solvents may be appropriatelychosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 0° C. to 40° C. for an hour to 4 hours, preferably, at10° C. to 30° C. for an hour to 3 hours, and more preferably, at 10° C.to 20° C. for an hour to 2 hours.

(2) In the next step, any one selected from the group consisting of thecompound represented by general formula (IX) and the compoundrepresented by general formula (X) in which the amino in the compoundrepresented by general formula (IX) is protected with R⁵ is subjected tothe ring-closing metathesis reaction followed by deprotecting R⁴ and, ifany, R⁵. Thus, the compound represented by general formula (XIV) isobtained.

(2-1) When the compound represented by general formula (X) is subjectedto the subsequent ring-closing metathesis reaction, the amino in thecompound represented by general formula (IX) is first protected with R⁵to give the compound represented by general formula (X).

Specifically, protection of the amino with R⁵ is performed as follows.The compound represented by general formula (IX) is reacted with acetylchloride, acetic anhydride, benzoyl chloride, di-t-butyl dicarbonate,benzyl chloroformate, etc. in the presence of a base such astriethylamine, diisopropylethylamine, pyridine and4-dimethylaminopyridine. More specifically, the compound represented bygeneral formula (X) wherein R⁵ is acetyl can be obtained by the processdescribed in SYNTHESIS EXAMPLES later described.

Specific examples of the solvents used in the reaction include pyridine,methylene chloride, tetrahydrofuran, toluene, etc. These solvents may beappropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 0° C. to 100° C. for an hour to 24 hours, preferably,at 20° C. to 70° C. for an hour to 12 hours, and more preferably, at 50°C. to 60° C. for an hour to 2 hours.

(2-2) Subsequently, any one selected from the group consisting of thecompound represented by general formula (IX) and the compoundrepresented by general formula (X) is subjected to the ring-closingmetathesis reaction to give a tricyclic aromatic compound. Morespecifically, the ring-closing metathesis reaction of the compoundrepresented by general formula (IX) or the compound represented bygeneral formula (X) is carried out in the presence of a Grubbs catalystor a Hoveyda-Grubbs catalyst to give the compound represented by generalformula (XI′) or the compound represented by general formula (XI).

In a preferred embodiment of the present invention, the compoundrepresented by general formula (X) is used as the compound provided forthe ring-closing metathesis. By using the compound represented bygeneral formula (X), the compound represented by general formula (XI)can be obtained in a good yield.

The Grubbs catalyst or the Hoveyda-Grubbs catalyst used in thering-closing metathesis reaction includes a Grubbs first generationcatalyst, a Grubbs second generation catalyst, a Hoveyda-Grubbs firstgeneration catalyst, a Hoveyda-Grubbs second generation catalyst, etc.,and preferably, a Hoveyda-Grubbs second generation catalyst.

Specific examples of the solvents used in the reaction includedichloroethane, dichloromethane, chloroform, trichloroethane,tetrachloroethane, benzene, toluene, xylene, monochlorobenzene,dichlorobenzene, hexane, heptane, octane, tetrahydrofuran, dioxane,diethyl ether, dibutyl ether, diisopropyl ether, dimethoxyethane, etc.These solvents may be appropriately chosen and used alone or as asolvent mixture thereof.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 25° C. to 110° C. for an hour to 48 hours, preferably,at 50° C. to 110° C. for an hour to 24 hours, and more preferably, at90° C. to 100° C. for 12 to 18 hours.

(2-3) After the compound represented by general formula (XI′) isobtained in (2-2) above, R⁴ in the compound represented by generalformula (XI′) is deprotected to give the compound represented by generalformula (XIV).

Specifically, R⁴ is deprotected as follows. The deprotection is effectedthrough the reaction with a Lewis acid or a Brønsted acid. Morespecifically, the deprotection of R⁴ which is methyl can be performed bythe procedures described in SYNTHESIS EXAMPLES later given.

Specific examples of the solvents used in the reaction include pyridine,tetrahydrofuran, toluene, dimethylformamide, dimethylsulfoxide, water,etc. These solvents may be appropriately chosen and used alone or as asolvent mixture thereof, or may not be used.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 0° C. to 300° C. for 0.1 to 60 hours, preferably, at30° C. to 250° C. for 0.5 to 12 hours, and more preferably, at 180° C.to 220° C. for 0.5 to an hour.

(2-4) On the other hand, when the compound represented by generalformula (XI) is obtained in (2-2) above, R⁴ in the compound representedby general formula (XI) is deprotected to give the compound representedby general formula (XIII)

Specifically, R⁴ is deprotected as follows. The deprotection is effectedby the reaction with a Lewis acid or a Brønsted acid. More specifically,the deprotection of R⁴ which is methyl can be performed by theprocedures described in SYNTHESIS EXAMPLES later given.

Specific examples of the solvents used in the reaction includedichloromethane, tetrahydrofuran, diethyl ether, dioxane, methanol, etc.These solvents may be appropriately chosen and used alone or as asolvent mixture thereof.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 0° C. to 50° C. for an hour to 24 hours, preferably,at 0° C. to 30° C. for 2 to 6 hours, and more preferably, at 0° C. to20° C. for 2 to 4 hours.

(2-5) Furthermore, R⁵ in the compound represented by general formula(XIII) is deprotected to give the compound represented by generalformula (XIV).

Specifically, R⁵ is deprotected as follows. The deprotection is effectedby heating in a solvent in the presence of a base such as sodiumhydrogencarbonate, potassium carbonate, etc. More specifically, thedeprotection of R⁵ which is acetyl can be performed by the proceduresdescribed in SYNTHESIS EXAMPLES later given.

Specific examples of the solvents used in the reaction include methanol,ethanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, water, etc.These solvents may be appropriately chosen and used alone or as asolvent mixture thereof. The reaction temperature and reaction time arenot particularly limited and are, e.g., at 40° C. to 100° C. for an hourto 24 hours, preferably, at 50° C. to 80° C. for 2 to 24 hours, and morepreferably, at 60° C. to 70° C. for 12 to 16 hours.

2.2. Process for Producing the Compound Represented by General Formula(II)

The compound represented by general formula (II) can be produced fromthe compound represented by general formula (VIII) as follows.

(1) The compound represented by general formula (XIV) is produced asdescribed above.

(2) Then, the compound represented by general formula (XIV) is reactedwith the compound represented by general formula (XV) to give thecompound represented by general formula (II).

The compound represented by general formula (XV) can be produced byknown processes. Specifically, the compound can be produced by theprocesses described in Adamczyk, M. et al., Synth. Commun., 32,3199-3205 (2002) or Baganz, H. & May, H.-J. Chem. Ber., 99, 3766-3770(1966) and Baganz, H. & May, H.-J. Angew. Chem., Int. Ed. Eng., 5, 420(1966), or modifications thereof. More specifically, the compoundrepresented by general formula (XV) can be produced by reacting asubstituted benzyl Grignard reagent with ethyl diethoxyacetate at a lowtemperature (−78° C.) or by reacting an α-diazo-α′-substituted phenylketone with tert-butyl hypochlorite in ethanol.

The solvent used in the reaction to obtain the compound represented bygeneral formula (II) is not particularly limited but various solvent canbe used.

Examples are dioxane, tetrahydrofuran, ether, methanol, ethanol, water,etc. They may be used alone or as an admixture thereof.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 0° C. to 200° C. for an hour to 96 hours, at roomtemperature to 150° C. for 3 to 72 hours, or at 60° C. to 120° C. for 6to 24 hours.

2.3. Process for Producing the Compound Represented by General Formula(VIII)

The compound represented by general formula (VIII) above can be producedfrom, e.g., the compound represented by general formula (IV), asfollows.

(1) In the first step, the Negishi coupling is performed on the compoundrepresented by general formula (IV) with an organic zinc compound.Specifically, the compound represented by general formula (IV) isreacted with R¹MgX and ZnCl₂ in the presence of a palladium catalyst togive the compound represented by general formula (V).

The compound represented by general formula (IV) is the compounddescribed in WO 2003/059893 and may be synthesized, e.g., by theprocedures described in SYNTHESIS EXAMPLES later given.

R¹MgCl used may be synthesized by known synthesis methods or may becommercially available.

Specific examples of the palladium catalyst used for the Negishicoupling include Pd(PPh₃)₄, PdCl₂(PPh₃)₂, Pd(OAc)₂,tris(dibenzylideneacetone)dipalladium(0),tris(dibenzylideneacetone)dipalladium(0) chloroform complex,bis(dibenzylideneacetone)palladium(0),bis(tri-t-butylphosphino)palladium(0),(1,1′-bis(diphenylphosphino)ferrocene)dichloropalladium(II), etc.

Specific examples of the solvents used in the reaction are benzene,toluene, xylene, N,N-dimethylformamide, tetrahydrofuran, diethyl ether,t-butyl methyl ether, 1,4-dioxane, etc. These solvents may beappropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 0° C. to 50° C. for an hour to 24 hours, preferably,at 10° C. to 40° C. for 2 to 24 hours, and more preferably, at 20° C. to30° C. for 6 to 16 hours.

(2) In the next step, the compound represented by general formula (V) isarylated at the 5-position selectively via the Suzuki-Miyaura couplingwith boronic acid. Specifically, the compound represented by generalformula (V) is reacted with the compound represented by general formula(VI) in the presence of a palladium catalyst and a base to give thecompound represented by general formula (VII).

The compound represented by general formula (VI) used may becommercially available or may be synthesized by known synthesis methods.Specific examples of the palladium catalyst used in the Suzuki-Miyauracoupling are allylpalladium(II) chloride (dimer), Pd(PPh₃)₄,PdCl₂(PPh₃)₂, Pd(OAc)₂, tris(dibenzylideneacetone)dipalladium(0),tris(dibenzylideneacetone)dipalladium(0) chloroform complex,bis(dibenzylideneacetone)palladium(0), etc.

Phosphine compounds may be added to these palladium catalyst, ifnecessary, to accelerate the reaction. Specific examples of thephosphine compounds are 2-(di-t-butylphosphino)-1-phenylindole,tri(t-butyl)phosphine, tricyclohexylphosphine,1-(N,N-dimethylaminomethyl)-2-(di-t-butylphosphino)ferrocene,1-(N,N-dibutylaminomethyl)-2-(di-t-butylphosphino)ferrocene,1-(methoxymethyl)-2-(di-t-butylphosphino)ferrocene,1,1′-bis(di-t-butylphosphino)ferrocene,2,2′-bis(di-t-butylphosphino)-1,1′-binaphthyl,2-methoxy-2′-(di-t-butylphosphino)-1,1′-binaphthyl,2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl, etc.

Specific examples of the base used in the reaction include sodiumcarbonate, potassium carbonate, cesium carbonate, sodiumhydrogencarbonate, sodium hydroxide, potassium hydroxide, bariumhydroxide, sodium ethoxide, sodium t-butoxide, sodium acetate,tripotassium phosphate, potassium fluoride, etc.

Specific examples of the solvents used in the reaction are benzene,toluene, xylene, N,N-dimethylformamide, tetrahydrofuran, diethyl ether,t-butyl methyl ether, 1,4-dioxane, methanol, ethanol, isopropyl alcohol,water, etc. These solvents may be appropriately chosen and used alone oras a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 0° C. to 50° C. for an hour to 24 hours, preferably,at 10° C. to 40° C. for 2 to 24 hours, and more preferably, at 20° C. to30° C. for 6 to 16 hours.

(3) In the next step, the 6-position of the pyrazine ring in thecompound represented by general formula (VII) is vinylated through theStille coupling with a vinyl compound. Specifically, the compoundrepresented by general formula (VII) is reacted with tributyl(vinyl)tinin the presence of a palladium catalyst to give the compound representedby general formula (VIII).

Tributyl(vinyl)tin used may be commercially available or may besynthesized by known methods.

Specific examples of the palladium catalyst used in the Stille couplingare Pd(PPh₃)₄, PdCl₂(PPh₃)₂, Pd(OAc)₂,tris(dibenzylideneacetone)dipalladium(0),tris(dibenzylideneacetone)dipalladium(0) chloroform complex,bis(dibenzylideneacetone)palladium(0), etc.

In order to accelerate the reaction, these palladium catalysts may beadded with tetra-n-butylammonium chloride, tetra-n-butylammoniumbromide, tetra-n-butylammonium iodide, etc., if necessary. Furthermore,polymerization inhibitors such as 2,6-di-tert-butyl-4-methylphenol(BHT), etc. may also be added to inhibit the formation of by-products.

Specific examples of the solvents used in the reaction are benzene,toluene, xylene, N,N-dimethylformamide, tetrahydrofuran, diethyl ether,t-butyl methyl ether, 1,4-dioxane, etc. These solvents may beappropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limitedand are, e.g., at 60° C. to 120° C. for an hour to 24 hours, preferably,at 80° C. to 110° C. for an hour to 12 hours, and more preferably, at100° C. to 110° C. for an hour to 2 hours.

3. Method for Producing the Calcium-Binding Photoprotein

The calcium-binding photoprotein of the present invention can beproduced or regenerated by contacting the compound represented bygeneral formula (II) (the v-coelenterazine compound of the presentinvention) with an apoprotein of the calcium-binding photoprotein togive the calcium-binding photoprotein.

As used herein, the term “contact” means that the v-coelenterazinecompound and an apoprotein of the calcium-binding photoprotein areallowed to be present in the same reaction system, and includes, forexample, the states that an apoprotein of the calcium-bindingphotoprotein is added to a container charged with the v-coelenterazinecompound, the v-coelenterazine compound is added to a container chargedwith an apoprotein of the calcium-binding photoprotein, thev-coelenterazine compound is mixed with an apoprotein of thecalcium-binding photoprotein, and the like. In one embodiment of thepresent invention, the contact is effected at a low temperature in thepresence of a reducing agent (e.g., mercaptoethanol, dithiothreitol,etc.) and oxygen. More specifically, the photoprotein of the presentinvention can be produced or regenerated by the methods described in,e.g., Shimomura, O. et al., Biochem. J, 251, 405-410 (1988), Shimomura,O. et al, Biochem. J, 261, 913-920 (1989), etc. The calcium-bindingphotoprotein of the present invention exists in the state that aperoxide of the v-coelenterazine compound produced from thev-coelenterazine compound and molecular oxygen and the apoprotein form acomplex in the presence of oxygen. When calcium ions bind to the complexdescribed above, the complex emits light to generate thev-coelenteramide compound as the oxide of the v-coelenterazine compoundand carbon dioxide. The complex described above is sometimes referred toas the “photoprotein of the present invention.”

Examples of the apoprotein used to produce the photoprotein of thepresent invention include apoaequorin, apoclytin-I, apoclytin-II,apobelin, apomitrocomin, apomineopsin, apobervoin, and the like. In someembodiments of the present invention, the apoprotein is apoaequorin,apobelin, apoclytin-I, apoclytin-II, mitrocomin, etc., e.g.,apoaequorin. These apoproteins may be obtained from natural sources orgenetically engineered. Furthermore, the amino acid sequence may also bemutated from the native sequence by gene recombination technology, aslong as the apoproteins are capable of producing the calcium-bindingphotoprotein.

The nucleotide sequences and amino acid sequences of the apoproteins ofphotoproteins obtained from the nature (native apoproteins) are asfollows. The nucleotide sequence and amino acid sequence of nativeapoaequorin are shown by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.The nucleotide sequence and amino acid sequence of native apoclytin-Iare shown by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The nucleotidesequence and amino acid sequence of native apoclytin-II are shown by SEQID NO: 5 and SEQ ID NO: 6, respectively. The nucleotide sequence andamino acid sequence of native apomitrocomin are shown by SEQ ID NO: 7and SEQ ID NO: 8, respectively. The nucleotide sequence and amino acidsequence of native apobelin are shown by SEQ ID NO: 9 and SEQ ID NO: 10,respectively. The nucleotide sequence and amino acid sequence of nativeapobervoin are shown by SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

The apoprotein mutated by recombinant technology is, for example, aprotein selected from the group consisting of (a) to (c) below:

(a) a protein comprising the amino acid sequence of native apoprotein inwhich 1 or more amino acids are deleted, substituted, inserted and/oradded, and having the activity or function of the apoprotein of thecalcium-binding photoprotein;

(b) a protein comprising an amino acid sequence which has 90% or morehomology to the amino acid sequence of native apoprotein, and having theactivity or function of the apoprotein of the calcium-bindingphotoprotein; and,

(c) a protein comprising an amino acid sequence encoded by apolynucleotide that hybridizes under stringent conditions to apolynucleotide comprising a nucleotide sequence complementary to thenucleotide sequence of native apoprotein, and having the activity orfunction of the apoprotein of the calcium-binding photoprotein.

Examples of the “native apoprotein” described above are apoaequorin,apoclytin-I, apoclytin-II, apobelin, apomitrocomin, apomineopsin,apobervoin, etc. In an embodiment of the present invention, theapoprotein is apoaequorin, apobelin, apoclytin-I, apoclytin-II,apobelin, apomitrocomin, etc., preferably apoaequorin. The amino acidsequences and nucleotide sequences of these native apoproteins are thesame as described above.

The term “activity or function of the apoprotein of the calcium-bindingphotoprotein” described above is used to mean, for example, the activityor function of the apoprotein which binds to a peroxide of thev-coelenterazine compound to produce the calcium-binding photoprotein.“The protein binds to a peroxide of the v-coelenterazine compound toproduce the calcium-binding photoprotein” specifically means not only(1) that the protein binds to the peroxide of v-coelenterazine compoundto produce the photoprotein, but also (2) that the protein is brought incontact with the v-coelenterazine compound in the presence of oxygen toproduce the photoprotein (complex) containing the protein and a peroxideof the v-coelenterazine compound. As used herein, the term “contact”means that the protein and the v-coelenterazine compound are allowed tobe present in the same reaction system, and includes, for example,addition of the protein to a container charged with the v-coelenterazinecompound, addition of the v-coelenterazine compound to a containercharged with the protein, mixing of the protein with thev-coelenterazine compound, and the like.

The “v-coelenterazine compound” refers to v-coelenterazine and as inv-coelenterazine, a compound capable of constituting as the apoproteinthe calcium-binding photoprotein such as aequorin, etc.(v-coelenterazine analogue), specifically, cf3-v-coelenterazine.

The range of “1 or more” in “the amino acid sequence in which 1 or moreamino acids are deleted, substituted, inserted and/or added” describedabove is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1. Ingeneral, the less the number of the amino acids deleted, substituted,inserted or added, the more preferable. In the deletion, substitution,insertion and addition of the amino acid residues described above, twoor more may occur concurrently. Such regions can be acquired by usingsite-directed mutagenesis described in “Sambrook J. et al., MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press (2001),” “Ausbel F. M. et al., Current Protocols inMolecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997),”“Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79,6409 (1982),” “Gene, 34, 315 (1985),” “Nuc. Acids. Res., 13, 4431(1985),” “Proc. Natl. Acad. Sci. USA, 82, 488 (1985),” etc.

The range of “90% or more” in the “amino acid sequence which has 90% ormore homology” is, for example, 90% or more, 91% or more, 92% or more,93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% ormore, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% ormore, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or99.9% or more. It is generally preferred for the numerical valueindicating the degree of homology to be higher. The homology betweenamino acid sequences or nucleotide sequences can be determined using asequencing program such as BLAST (see, e.g., Altzshul S. F. et al., J.Mol. Biol., 215, 403 (1990), etc.) or the like. When BLAST is used, thedefault parameters for the respective programs are used.

The “polynucleotide that hybridizes under stringent conditions”described above refers to a polynucleotide (e.g., DNA) which is obtainedby, for example, colony hybridization, plaque hybridization, Southernhybridization, etc., using as a probe all or part of a polynucleotidecomprising a nucleotide sequence complementary to the nucleotidesequence of native apoprotein or a polynucleotide encoding the aminoacid sequence of native apoprotein. Specific examples include apolynucleotide which can be identified by performing hybridization at65° C. in the presence of 0.7 to 1.0 mol/L NaCl using a filter on whichthe polynucleotide from a colony or plaque is immobilized, then washingthe filter under 65° C. conditions with an SSC (saline-sodium citrate)solution having a concentration of 0.1 to 2 times (a 1×SSC solution iscomposed of 150 mmol/L of sodium chloride and 15 mmol/L of sodiumcitrate).

Hybridization may be performed in accordance with modifications of themethods described in textbooks of experiment, e.g., Sambrook J. et al.,Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press (2001), Ausbel F. M. et al., Current Protocolsin Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997),Glover D. M. and Hames B. D., DNA Cloning 1: Core Techniques, Apractical Approach, Second Edition, Oxford University Press (1995), etc.

As used herein, the term “stringent conditions” may refer to lessstringent conditions, moderately stringent conditions and highlystringent conditions. The “less stringent conditions” are, for example,the conditions under 5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS and50% (v/v) formamide at 32° C. The “moderately stringent conditions” are,for example, the conditions under 5×SSC, 5×Denhardt's solution, 0.5%(w/v) SDS and 50% (v/v) formamide at 42° C. The “highly stringentconditions” are, for example, the conditions under 5×SSC, 5×Denhardt'ssolution, 0.5% (w/v) SDS and 50% (v/v) formamide at 50° C. The morestringent the conditions are, the higher the complementarity requiredfor double strand formation. Specifically, for example, under theseconditions, a polynucleotide (e.g., DNA) of higher homology is expectedto be obtained efficiently at higher temperatures, although multiplefactors are involved in hybridization stringency, including temperature,probe concentration, probe length, ionic strength, time and baseconcentration; those skilled in the art may appropriately select thesefactors to realize a similar stringency.

When a commercially available kit is used for the hybridization, forexample, AlkPhos Direct Labeling Reagents (manufactured by AmershamPharmacia) can be used. According to the protocol attached to the kit inthis case, incubation with a labeled probe is performed overnight, themembrane is then washed with a primary wash buffer containing 0.1% (w/v)SDS at 55° C., and finally the hybridized DNA can be detected.

Other hybridizable polynucleotides include, as calculated by asequencing program such as BLAST or the like using the defaultparameters, DNAs having homology of approximately 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more,90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% ormore, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, or99.9% or more, to the polynucleotide encoding the amino acid sequence ofthe apoprotein. The homology of an amino acid sequence or a nucleotidesequence can be determined using the method described hereinabove.

Examples of the recombinant apoprotein which can be used in the presentinvention include recombinant aequorin described in Shimomura, O. andInouye, S. Protein Express. Purif. (1999) 16: 91-95, recombinantclytin-I described in Inouye, S. and Sahara, Y. Protein Express. Purif.(2007) 53: 384-389, recombinant clytin-II described in Inouye, S. J.Biochem. (2008) 143: 711-717, and the like.

The calcium-binding photoprotein thus produced may be further purified.Purification of the calcium-binding photoprotein can be performed in aconventional manner of separation/purification. Theseparation/purification includes, for example, precipitation withammonium sulfate, gel filtration chromatography, ion exchangechromatography, affinity chromatography, reversed phase high performanceliquid chromatography, dialysis, ultrafiltration, etc., alone or in anappropriate combination of these techniques.

4. Use of v-Coelenterazine Compound of the Invention or Photoprotein ofthe Invention

(1) Use as Luminescence Substrate

The v-coelenterazine compound in some embodiments of the presentinvention emits light by the action of an emission-catalyzing enzyme andcan be used as a luminescence substrate. Therefore, the presentinvention provides a method for emitting light which comprisescontacting the v-coelenterazine compound of the present invention withan emission-catalyzing enzyme. As used herein, the term “contact” meansthat the v-coelenterazine compound of the present invention and anemission-catalyzing enzyme are allowed to be present in the samereaction system, and includes, for example, the states that theemission-catalyzing enzyme is added to a container charged with thev-coelenterazine compound, the v-coelenterazine compound is added to acontainer charged with the emission-catalyzing enzyme, thev-coelenterazine compound is mixed with the emission-catalyzing enzyme,and the like.

The emission-catalyzing enzyme used in the method of the presentinvention for emitting light is, for example, a luciferase derived fromOplophorus sp., e.g., Oplophorus grachlorostris (Oplophorus luciferase),a luciferase derived from Gaussia sp., e.g., Gaussia princeps (Gaussialuciferase), a luciferase derived from Renilla sp., e.g., Renillareniformis, or Renilla muelleri (Renilla luciferase), a luciferasederived from Pleuromamma sp. (Pleuromamma luciferase) or a luciferasederived from Metridia Tonga (Metridia luciferase).

The emission-catalyzing enzyme used for the method of the presentinvention for emitting light may be mutants of these luciferases.Examples of the mutants include mutants of Renilla luciferase describedin Loening et al. Protein Eng. Des. Sel. (2006) 19, 391-400, mutants ofRenilla luciferase described in Loening et al. Nature methods (2007) 4,641-643, mutants of Renilla luciferase described in Woo et al. ProteinSci. (2008) 17, 725-735, etc.

These emission-catalyzing enzymes can be prepared by the methoddescribed in, e.g., Shimomura et al. (1988) Biochem. J. 251, 405-410,Shimomura et al. (1989) Biochem. J. 261, 913-920, or Shimomura et al.(1990) Biochem. J. 270, 309-312, or modifications thereof.Alternatively, various types of the enzymes are commercially availablefrom JNC Corporation (former Chisso Corporation), Wako Pure Chemical,Promega Inc., etc. and these enzymes commercially available may also beemployed for the method of the present invention for emitting light.Among the Renilla luciferase, the nucleotide sequence and amino acidsequence of Renilla reniformis-derived luciferase are shown by SEQ IDNO: 13 and SEQ ID NO: 14, respectively. The nucleotide sequence andamino acid sequence of Oplophorus grachlorostris-derived luciferaseamong the Oplophorus luciferase are shown by SEQ ID NO: 15 and SEQ IDNO: 16, respectively. The nucleotide sequence and amino acid sequence ofGaussia princeps-derived luciferase in the Gaussia luciferase are shownby SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

The nucleotide sequence and amino acid sequence of Renilla luciferasemutant described in Inouye & Shimomura, Biochem. Biophys. Res. Commun.,233 (1997) 349-353 are shown by SEQ ID NO: 19 and SEQ ID NO: 20,respectively.

In one embodiment of the present invention, Renilla reniformis-derivedluciferase, e.g., a protein comprising a polypeptide consisting of theamino acid sequence of SEQ ID NO: 14 is used as the emission-catalyzingenzyme.

In another embodiment of the present invention, a mutant of Renillaluciferase, e.g., a protein comprising a polypeptide consisting of theamino acid sequence of SEQ ID NO: 20 is used as the emission-catalyzingenzyme. In some embodiments of the present invention, the maximumemission wavelength can be shifted toward the longer wavelength siderelative to Renilla reniformis-derived luciferase by using the mutant ofRenilla luciferase as the emission-catalyzing enzyme.

Light emits by contacting these emission-catalyzing enzymes with thev-coelenterazine compound in some embodiments of the present invention.The emission time is usually 0.01 to an hour. However, the emission timemay be prolonged or further shortened by selecting conditions.

(2) Detection or Quantification of Calcium Ions

The photoprotein of the present invention obtained as described above isa non-covalent complex of an apoprotein and a peroxide of thev-coelenterazine compound formed from the v-coelenterazine compound andmolecular oxygen and is a photoprotein (holoprotein) that emits light bythe action of calcium ions. Therefore, the photoprotein of the inventioncan be used for the detection or quantification of calcium ions.

The detection or quantification of calcium ions can be performed, forexample, by adding a sample solution directly to a solution of thephotoprotein and measuring the luminescence generated. Alternatively,calcium ions can also be detected or quantified by adding a solution ofthe photoprotein to a sample solution and measuring the luminescencegenerated. The photoprotein above may be formed by previously contactingan aqueous apoprotein solution with the v-coelenterazine compound of thepresent invention prior to its addition to the assay system for thedetection or quantification of calcium ions and the resultingphotoprotein may be provided for use. The photoprotein comprising anapoprotein and a peroxide of the v-coelenterazine compound may also beformed in the assay system by contacting the apoprotein with thev-coelenterazine compound. The photoprotein formed is a complex(photoprotein) of the apoprotein and the peroxide of thev-coelenterazine compound of the invention. The above complex (i.e., thephotoprotein of the present invention) emits light dependently on thecalcium ion concentration.

The detection or quantification of calcium ions can be performed bymeasuring the luminescence of the photoprotein of the invention throughthe action of calcium ions, using a luminometer. Luminometers which maybe used include commercially available instruments, such as a Centro LB960 (manufactured by Berthold, Inc.), etc. The calcium ion concentrationcan be quantitatively determined by preparing a luminescence standardcurve for known calcium ion concentrations using the photoprotein.

The v-coelenterazine compound of the present invention may also be usedfor the detection of changes in the intracellular calcium ionconcentration under the physiological conditions by preparing thephotoprotein comprising an apoprotein and a peroxide of thev-coelenterazine compound and injecting the photoprotein directly intocells by means of microinjection, etc.

The v-coelenterazine compound of the present invention may also be usedto produce the photoprotein, which is performed, in addition to theinjection using techniques such as microinjection, by intracellularlyexpressing a gene for the apoprotein (a polynucleotide encoding theapoprotein) to produce the protein in the cells and adding thev-coelenterazine compound of the present invention to the resultingapoprotein from the external cells.

Using the photoprotein of the present invention thus introduced intocells or produced in cells, changes in the intracellular calcium ionconcentration caused by external stimulation (e.g., stimulation withreceptor-associated drugs, etc.) can also be determined.

(3) Use as Reporter Protein, etc. Utilizing Luminescence

The photoprotein of the present invention can also be used as a reporterprotein to determine the transcription activity of a promoter, etc. Apolynucleotide encoding an apoprotein is fused to a target promoter orother expression control sequence (e.g., an enhancer, etc.) to constructa vector. The vector described above is transfected to a host cell, andthe v-coelenterazine compound of the present invention is brought incontact with the cell. By detecting the luminescence from thephotoprotein of the present invention, the activity of the targetpromoter or other expression control sequence can be determined. As usedherein, the term “contact” means that a host cell and thev-coelenterazine compound are allowed to be present in the same culturesystem/reaction system, and includes, for example, addition of thev-coelenterazine compound to a culture container charged with a hostcell, mixing of a host cell with the v-coelenterazine compound, cultureof a host cell in the presence of the v-coelenterazine compound, and thelike.

The v-coelenterazine compound of the present invention can be used todetermine the transcription activity of a promoter, etc. For example, apolynucleotide encoding the emission-catalyzing enzyme is fused to atarget promoter or other expression control sequence (e.g., an enhancer,etc.) to construct a vector. The vector described above is transfectedto a host cell, and the v-coelenterazine compound of the presentinvention is contacted with the cell. By detecting the luminescence fromthe v-coelenterazine compound of the present invention, the activity ofthe target promoter or other expression control sequence can bedetermined. As used herein, the term “contact” means that a host celland the v-coelenterazine compound of the present invention are allowedto be present in the same culture system/reaction system, and includes,for example, addition of the v-coelenterazine compound to a culturecontainer charged with a host cell, mixing of a host cell with thev-coelenterazine compound, culture of a host cell in the presence of thev-coelenterazine compound, and the like. The emission-catalyzing enzymeis described above and is at least one luciferase selected from thegroup consisting of Renilla luciferase, Oplophorus luciferase, Gaussialuciferase and mutants thereof, and preferably, Renilla luciferasemutants.

The present invention further provides a kit used for the measurement oftranscription activity of a promoter, etc. In some embodiments of thepresent invention, the kit comprises the v-coelenterazine compound ofthe present invention and the emission-catalyzing enzyme. In anotherembodiment of the present invention, the kit comprises the photoproteinof the present invention and the coelenterazine compound. Reagent suchas the coelenterazine compound, the emission-catalyzing enzyme, etc. maybe dissolved in a suitable solvent and prepared to be suitable forstorage. The solvent which may be used is at least one selected from thegroup consisting of water, ethanol, various buffer solutions, and thelike. The kit may additionally comprise, if necessary, at least oneselected from the group consisting of a container designed therefor,other necessary accessories and an instruction manual, and the like.

(4) Use as Detection Marker, etc. Utilizing Luminescence

The photoprotein of the present invention can be used as a marker fordetection by luminescence. The detection marker of the present inventioncan be used to detect a target substance in, e.g., immunoassay,hybridization assay, etc. The photoprotein of the present invention canbe used in the form bound to a target substance (protein, nucleic acid,etc.) in a conventional manner including chemical modification.Detection methods using such a detection marker can be performed in aconventional manner. The detection marker of the present invention canalso be used to determine the distribution of a target substance byexpressing the marker, e.g., as a fusion protein of the apoprotein andthe target substance, then inserting the fusion protein into cells bymeans of microinjection or the like and contacting them with thev-coelenterazine compound of the present invention thereby to producethe photoprotein of the present invention. As used herein, the term“contact” means that cells and the v-coelenterazine compound of thepresent invention are allowed to be present in the same culturesystem/reaction system, and includes, for example, addition of thev-coelenterazine compound of the present invention to a culturecontainer charged with cells, mixing of cells with the v-coelenterazinecompound of the present invention, culture of host cells in the presenceof the v-coelenterazine compound of the present invention, and the like.

The present invention further provides a method for detection of atarget substance in, e.g., immunoassay, hybridization assay, etc., whichcomprises using the v-coelenterazine compound and theemission-catalyzing enzyme. In this case, the emission-catalyzing enzymecan be used in the form bound to the target substance (protein, nucleicacid, etc.) in a conventional manner, including chemical modification.Such a detection method using the detection marker can be performed in aconventional manner. The detection marker can also be used to determinethe distribution of the target substance by expressing the marker, e.g.,as a fusion protein of the emission-catalyzing enzyme and the targetsubstance, then inserting the fusion protein into cells by means ofmicroinjection or the like and contacting it with the v-coelenterazinecompound of the present invention. As used herein, the term “contact”means that a cell and the v-coelenterazine compound of the presentinvention are allowed to be present in the same culture system/reactionsystem, and includes, for example, addition of the v-coelenterazinecompound of the present invention to a culture container charged with acell, mixing of a cell with the v-coelenterazine compound of the presentinvention, culture of a host cell in the presence of thev-coelenterazine compound of the present invention, and the like. Theemission-catalyzing enzyme is the same as described above and is, forexample, at least one luciferase selected from the group consisting ofRenilla luciferase, Oplophorus luciferase and Gaussia luciferase.

The distribution of these target substances, etc. can be determined by amethod for detection such as luminescence imaging, etc. The apoproteinmay also be used after its expression in cells, in addition to theinsertion into cells by means of microinjection, etc.

The present invention further provides a kit used for the detection of atarget substance in an immunoassay, hybridization assay, etc. The kit insome embodiments of the present invention comprises the photoprotein ofthe present invention and the coelenterazine compound. The kit inanother embodiment of the present invention comprises thev-coelenterazine compound of the present invention and theemission-catalyzing enzyme. Reagent such as the coelenterazine compound,the emission-catalyzing enzyme, etc. may be dissolved in a suitablesolvent and prepared to be suitable for storage. The solvent which maybe used is at least one selected from the group consisting of water,ethanol, various buffer solutions, and the like. The kit mayadditionally comprise, if necessary, at least one selected from thegroup consisting of a container designed therefor, other necessaryaccessories and an instruction manual, and the like.

(5) Material for Amusement Supplies

The complex (photoprotein of the present invention) comprising theapoprotein and a peroxide of the v-coelenterazine compound of thepresent invention emits light only by binding to a trace of calciumions. The photoprotein of the present invention can thus be preferablyused as a luminescence material for amusement supplies. Examples of suchamusement supplies are luminescent soap bubbles, luminescent ice,luminescent candies, luminescent color paints, etc. The amusementsupplies of the present invention can be prepared in a conventionalmanner.

(6) Bioluminescence Resonance Energy Transfer (BRET) Method

In some embodiments of the present invention, the v-coelenterazinecompound emits light by the action of the emission-catalyzing enzyme asdescribed above, and can be used for analysis methods including ananalysis of physiological functions, an analysis of (or assay for)enzyme activities, etc., based on the principle of intermolecularinteraction by the bioluminescence resonance energy transfer (BRET)method. The photoprotein of the present invention can also be used foranalyses including an analysis of biological functions, an assay forenzyme activities, etc., based on the principle of intermolecularinteraction by the bioluminescence resonance energy transfer (BRET)method.

For example, using the v-coelenterazine compound and theemission-catalyzing enzyme in some embodiments of the present inventionas donor proteins and as an acceptor protein an organic compound or afluorescent protein, the interaction between the proteins can bedetected by generating bioluminescence resonance energy transfer (BRET)between them. As used herein, the emission-catalyzing enzyme is the sameas given before and is, for example, at least one luciferase selectedfrom the group consisting of Renilla luciferase, Oplophorus luciferaseand Gaussia luciferase.

Alternatively, using the photoprotein of the invention as a donorprotein and an organic compound or a fluorescent protein as an acceptorprotein, the interaction between the proteins can be detected bygenerating bioluminescence resonance energy transfer (BRET) betweenthem. In a certain embodiment of the present invention, the organiccompound used as an acceptor protein is Hoechist 3342, Indo-1, DAP1,etc. In another embodiment of the present invention, the fluorescentprotein used as an acceptor protein is a green fluorescent protein(GFP), a blue fluorescent protein (BFP), a mutant GFP fluorescentprotein, phycobilin, etc. In a preferred embodiment of the presentinvention, the physiological functions to be analyzed include an orphanreceptor (in particular, G-protein conjugated receptor), apoptosis,transcription regulation, etc. by gene expression. Still in a preferredembodiment of the present invention, the enzyme to be analyzed isprotease, esterase, kinase, etc.

Analysis of the physiological functions by the BRET method may beperformed by known methods, for example, by modifications of the methodsdescribed in Biochem. J. 2005, 385, 625-637, Expert Opin. Ther. Targets,2007 11: 541-556, etc. Assay for the enzyme activities may also beperformed by known methods, for example, by modifications of the methodsdescribed in Nat. Methods 2006, 3:165-174, Biotechnol. J. 2008, 3:311-324, etc.

The present invention further provides a kit used for the analysismethods described above. The kit comprises the v-coelenterazine compoundof the present invention, the emission-catalyzing enzyme and the organiccompound and/or the fluorescent protein. Reagents such as thev-coelenterazine compounds, the emission-catalyzing enzymes, organiccompounds, fluorescent proteins, etc. may be dissolved in a suitablesolvent and prepared to be suitable for storage. The solvent which maybe used is at least one selected from the group consisting of water,ethanol, various buffer solutions, and the like. The kit mayadditionally comprise, if necessary, at least one selected from thegroup consisting of a container designed therefor, other necessaryaccessories and an instruction manual, and the like.

All literatures and publications mentioned in this specification areherein incorporated in their entirety by reference into thespecification, irrespective of their purposes. The disclosure of thespecification, the claims, abstract and drawings of Japanese ApplicationJP2011-050487 filed on Mar. 8, 2011, based upon which the presentapplication claims the benefit of priority, are entirely incorporatedherein by reference.

The objects, characteristics and advantages of the present invention aswell as the idea thereof are apparent to those skilled in the art fromthe descriptions given herein, and those skilled in the art can easilyimplement the present invention. It is to be understood that the bestmode to carry out the invention and specific examples are to be taken aspreferred embodiments of the present invention. These descriptions areonly for illustrative and explanatory purposes and are not intended torestrict the invention thereto. It is apparent to those skilled in theart that various modifications may be made based on the descriptionsgiven herein within the intent and scope of the present inventiondisclosed herein.

EXAMPLES

Hereinafter, the present invention will be illustrated more specificallywith reference to SYNTHESIS EXAMPLES and EXAMPLES, but it should beunderstood that the invention is not deemed to be limited to theseSYNTHESIS EXAMPLES and EXAMPLES.

Synthesis Examples Overview

The process for producing the v-coelenterazine compound illustrativelyshown in these SYNTHESIS EXAMPLES is a highly flexible and highlyefficient process which is applicable to the production of variousv-coelenterazine analogues.

More specifically, in these SYNTHESIS EXAMPLES, commercially available2-chloro-6-aminopyrazine 3 was first dibrominated to synthesize knownCompound 4 (WO 2003/059893). The compound was subjected to the Negishicoupling with benzyl zinc reagent to introduce benzyl into Compound 4,whereby Compound 5 could be obtained. Next, the Suzuki-Miyaura couplingwas performed between Compound 5 and boronic acid 6 so that Compound 5was arylated at the 5-position selectively to give Compound 7. Then, the6-position of Compound 7 was vinylated through the Stille coupling withvinyl tin to give Compound 8 successfully. It is considered that therespective substituents could be introduced with high positionselectivity by the effect of unprotected amino group in these 3 types ofthe coupling reactions.

The formyl group of Compound 8 obtained was further methylated to giveCompound 9. After the amino group in Compound 9 was protected withacetyl, the ring-closing metathesis reaction was performed using aHoveyda-Grubbs second generation catalyst, whereby tricyclic aromaticCompound II could be efficiently obtained. In this case, though theyield was low, the ring-closing metathesis reaction could proceed togive the corresponding cyclic product 12 from Compound 9 with the aminobeing unprotected. However, it was found that the ring closing reactionproceeded more efficiently with an acyl-protected compound.

Subsequently, the methyl group in Compound II was removed by borontribromide. The resulting Compound 13 was heated in methanol in thepresence of a small quantity of sodium hydrogencarbonate fordeprotection of acetyl to give v-coelenteramine 4. For removal of themethyl group in Compound 12 wherein the amino group is not protectedwith acetyl group, boron tribromide could not be used but by heatingwith pyridine hydrochloride, v-coelenteramine 14 could be obtained whilethe yield was low.

Finally, v-coelenteramine 14 was condensed and cyclized with theketacetal 15. Thus, v-CTZ (2) could be synthesized.

This synthetic route is as short as 8 to 10 steps from the commerciallyavailable compound. In addition, the stability is expected to beimproved by varying the substituent on the benzene ring of the ketacetalused in the final step, and a variety of v-CTZ analogues can besynthesized in a simple manner (cf, e.g., the equation below).

In the future it is expected that v-CTZ compounds suitable for diverseapplications including near-infra red luminescent imaging can be newlyproduced by using this process for synthesis.

Materials and Methods (1) Chromatography

Thin layer chromatography (TLC) for analysis was performed on a glassplate (MERCK 5715, silica gel 60 F₂₅₄) previously coated with silicagel. The spots were detected under a UV lamp (254 nm or 365 nm) byadsorbing iodine, dipping in an aqueous anisaldehyde solution andcharring on a hot plate.

For preparative flush column chromatography, silica gels (Kanto ChemicalCo., Inc., 37563-85, silica gel 60 N (spherical, neutral), particle size45-50 μm or Kanto Chemical Co., Inc., 37565-85, silica gel 60 N(spherical, neutral), particle size 63-210 μm) were used. Forpurification of the CTZ analogues, however, silica gels (Kanto ChemicalCo., Inc., 37562-79, silica gel 60 N (spherical), particle size 40-50 μmor Kanto Chemical Co., Inc., 37558-79, silica gel 60 N (spherical),particle size 100-210 μm) were used.

(2) Nuclear Magnetic Resonance (NMR) Spectrum

¹H Nuclear magnetic resonance (NMR) spectrum (400 MHz) was determined onan AVANCE 400 or AVANCE 500 nuclear magnetic resonance apparatusmanufactured by Bruker, Inc. Chemical shifts (δ) were expressed asvalues relative to the peaks from tetramethylsilane ((CH₃)₄Si) (measuredin CDCl₃; 0 ppm) or the peaks from non-deuterated solvent for analysis(measured in CD₃OD; 3.31 ppm; measured in DMSO-d₆; 2.49 ppm) as aninternal standard. Abbreviations s, d and m used for the signalsplitting patterns represent singlet, doublet and multiplet,respectively.

¹³C Nuclear magnetic resonance spectrum (100 MHz) was determined by aAVANCE 400 nuclear magnetic resonance apparatus manufactured by Bruker,Inc. Chemical shifts (δ) were expressed as values relative to the peaksfrom carbon in the solvent for analysis (measured in CDCl₃; 77.0 ppm,measured in CD₃OD; 49.0 ppm) as an internal standard.

(3) Infrared Absorption (IR) Spectrum

IR spectra were determined by the diffuse reflection method using aSHIMAZU IR Prestige-21 spectrophotometer equipped with DRS-8000,manufactured by Shimadzu Corporation.

(4) Mass Spectrometry

High resolution mass spectrometry (HRMS) was performed by theelectrospray ionization method (ESI) with a Bruker micrOTOF manufacturedby Bruker, Inc.

(5) Chemical Reagent

The reagents were used without further treatment unless otherwiseindicated. The solvents for the reactions, extractions andchromatography were acetonitrile, ethyl acetate, n-hexane, anhydroustetrahydrofuran (THF), toluene, anhydrous pyridine, 1,2-dichloroethane,anhydrous dichloromethane, methanol and ethanol, all commerciallyavailable, and used without further treatment. Unless otherwiseindicated, mixing ratios in the solvent mixtures used are based on“v/v.”

The reaction reagents below were used. 2-Amino-6-chloropyrazine (Cat.No. 1650) purchased from Matrix Scientific, N-bromosuccinimide (Cat. No.025-07235), 2-formyl-4-methoxyphenylboronic acid (Cat. No. 328-99903),potassium fluoride (Cat. No. 169-03765), tetra-n-butylammonium chloride(Cat. No. T0054), methyltriphenylphosphonium bromide (Cat. No.138-11961), 4-(dimethylamino)pyridine (Cat. No. 042-19212), acetylchloride (Cat. No. 011-0053), conc. hydrochloric acid (Cat. No.080-01066) and pyridinium hydrochloride (Cat. No. 163-11931) purchasedfrom Wako Pure Chemical Co., Ltd., zinc chloride (1.0 M in diethylether) (Cat. No. 276839), benzyl magnesium chloride (2.0 M in THF) (Cat.No. 225916), dichlorobis(triphenylphosphine) palladium(II) (Cat. No.412740), 2-(di-tert-butylphosphino)-1-phenylindole (Cat. No. 672343),tributyl(vinyl)tin (Cat. No. 271438), Hoveyda-Grubbs second generationcatalyst (Cat. No. 569755) and boron tribromide (1.0 M indichloromethane) (Cat. No. 211222) purchased from Aldrich, Inc., allylpalladium(II) chloride dimer (Cat. No. A1479) purchased from TokyoChemical Industry Co., Ltd., n-butyl lithium (1.65 M in n-hexane (Cat.No. 04937-25) purchased from Kanto Chemical Co., Inc.; were used in thereactions without further treatment.

Synthesis Example 1 2-Amino-3,5-dibromo-6-chloropyrazine (4)

To a solution of 2-amino-6-chloropyrazine (3) (8.00 g, 61.8 mmol) inacetonitrile (80 mL) was gradually added N-bromosuccinimide (NBS) (27.5g, 155 mmol) at 0° C. After elevating to room temperature, the mixturewas stirred overnight (18 hours). To the mixture was added water and theproduct was extracted with diethyl ether (×3). The combined organicextract was washed successively with water (×1) and brine (×1), followedby drying over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was purified by silicagel flash column chromatography (n-hexane/ethyl acetate=3/1) to give2-amino-3,5-dibromo-6-chloropyrazine (4) (16.8 g, 58.5 mmol, 94.7%) as ayellow solid. TLC R_(f)=0.31 (n-hexane/ethyl acetate=4/1); ¹H NMR (500MHz, CDCl₃) δ 5.14 (s, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 120.7, 122.0,146.1, 151.0.

Synthesis Example 2 2-Amino-3-benzyl-5-bromo-6-chloropyrazine (5)

Under an argon atmosphere, to anhydrous THF (40 mL) was added zincchloride (1.0 M in diethyl ether) (23.7 mL, 23.7 mmol), and to themixture was added benzyl magnesium chloride (2.0 M in THF) (10.4 mL,20.8 mmol). After stirring at room temperature for 30 minutes, to themixture were successively addeddichlorobis(triphenylphosphine)palladium(II) (488 mg, 695 μmol) and2-amino-3,5-dibromo-6-chloropyrazine (4) (4.00 g, 13.9 mmol) at roomtemperature. The mixture was stirred overnight (15 hours and a half) atroom temperature as it was. After to the mixture was added water, theproduct was extracted with ethyl acetate (x 3). The combined organicextract was washed successively with water (×1) and brine (×1), followedby drying over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was purified by silicagel flash column chromatography (n-hexane/ethyl acetate=5/1) to give2-amino-3-benzyl-5-bromo-6-chloropyrazine (5) (3.64 g, 12.2 mmol, 87.6%)as a yellow solid. TLC R_(f)=0.25 (n-hexane/ethyl acetate=4/1); ¹H NMR(500 MHz, CDCl₃) δ 4.08 (s, 2H), 4.50 (s, 2H), 7.19-7.24 (m, 2H),7.27-7.37 (m, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 40.1, 123.8, 127.5, 128.4,129.3, 135.3, 139.5, 144.9, 151.3.

Synthesis Example 32-(5-Amino-6-benzyl-3-chloropyrazin-2-yl)-5-methoxybenzaldehyde (7)

Under an argon atmosphere, to a solution of allylpalladium(II) chloridedimer (160 mg, 437 μmol) in anhydrous THF (30 mL) was added2-(di-tert-butylphosphino)-1-phenylindole (295 mg, 874 μmol) at roomtemperature, followed by stirring at room temperature for 10 minutes.Subsequently, to the mixture were successively added2-amino-3-benzyl-5-bromo-6-chloropyrazine (5) (2.60 g, 8.74 mmol),2-formyl-4-methoxyphenylboronic acid (6) (3.14 g, 17.4 mmol), potassiumfluoride (2.60 g, 44.8 mmol) and water (160 μL, 8.88 mmol) at roomtemperature. The mixture was stirred overnight (14 hours) at roomtemperature without further treatment. After to this was added water,the product was extracted with ethyl acetate (×3). The combined organicextract was washed successively with water (×1) and brine (×1), anddried over anhydrous sodium sulfate. After filtration and concentrationunder reduced pressure, the residue was purified by silica gel flashcolumn chromatography (n-hexane/ethyl acetate=3/1) to give2-(5-amino-6-benzyl-3-chloropyrazin-2-yl)-5-methoxybenzaldehyde (7)(2.37 g, 6.70 mmol, 76.9%) as a reddish brown solid. TLC R_(f)=0.27(n-hexane/ethyl acetate=3/1); ¹H NMR (500 MHz, CDCl₃) δ 3.92 (s, 3H),4.13 (s, 2H), 4.66 (s, 2H), 7.21-7.25 (m, 3H), 7.27-7.31 (m, 1H),7.32-7.37 (m, 2H), 7.51-7.55 (m, 2H), 9.92 (s, 1H); ¹³C NMR (126 MHz,CDCl₃) δ 40.4, 55.7, 111.2, 120.7, 127.4, 128.4 (2C), 129.2 (2C), 132.5,132.7, 135.7, 135.8, 137.8, 139.1, 144.1, 151.5, 160.0, 191.0.

Synthesis Example 42-(5-Amino-6-benzyl-3-vinylpyrazin-2-yl)-5-methoxybenzaldehyde (8)

Under an argon atmosphere, to a solution of2-(5-amino-6-benzyl-3-chloropyrazin-2-yl)-5-methoxybenzaldehyde (7)(2.28 g, 6.44 mmol) in deaerated toluene (30 mL) were successively addeddichlorobis(triphenylphosphine)palladium(II) (226 mg, 322 μmol),tributyl(vinyl)tin (3.75 mL, 12.8 mmol) and tetra-n-butylammoniumchloride (3.50 g, 12.6 mmol) at room temperature. The mixture was heatedto reflux for 1.5 hours (oil bath temperature at 110° C.). After coolingto room temperature, to the mixture was added water and the product wasextracted with ethyl acetate (×3). The combined organic extract waswashed successively with water (×1) and brine (×1) followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by silica gel flash columnchromatography (silica gel mixed with 10 wt % potassium fluoride,n-hexane/ethyl acetate=3/1) to give2-(5-amino-6-benzyl-3-vinylpyrazin-2-yl)-5-methoxybenzaldehyde (8) (1.24g, 3.59 mmol, 55.5%) as a reddish brown oily substance. TLC R_(f)=0.27(n-hexane/ethyl acetate=3/1); ¹H NMR. (500 MHz, CDCl₃) δ 3.92 (s, 3H),4.15 (s, 2H), 4.50 (s, 2H), 5.39 (dd, 1H, J=2.0, 11.0 Hz), 6.33 (dd, 1H,J=2.0, 17.0 Hz), 6.57 (dd, 1H, J=11.0, 17.0 Hz), 7.20-7.28 (m, 4H),7.30-7.36 (m, 2H), 7.41 (d, 1H, J=8.5 Hz), 7.55 (d, 1H, J=3.0 Hz), 9.88(s, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 41.0, 55.7, 110.6, 120.5, 121.0,127.2, 128.5 (2C), 129.1 (2C), 132.3, 132.9, 134.4, 136.1, 136.5, 138.7,140.2, 145.2, 151.5, 159.8, 191.5.

Synthesis Example 52-Amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine (9)

Under an argon atmosphere, to a suspension of methyltriphenylphosphoniumbromide (1.29 g, 3.61 mmol) in anhydrous THF (5 mL) was graduallydropwise added n-butyl lithium (1.65 M in n-hexane) (1.70 mL, 2.81 mmol)at 0° C. The mixture was stirred at 0° C. for 15 minutes as it was. Tothe mixture was added2-(5-amino-6-benzyl-3-vinylpyrazin-2-yl)-5-methoxybenzaldehyde (8) (871mg, 2.52 mmol) at 0° C. After elevating to room temperature, stirringwas continued for 1.5 hours. After to this was added water, the productwas extracted with ethyl acetate (×3). The combined organic extract waswashed successively with water (×1) and brine (×1), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by silica gel flash columnchromatography (n-hexane/ethyl acetate=3/1) to give2-amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine (9) (570mg, 1.66 mmol, 65.8%) as a white solid. TLC R_(f)=0.37 (n-hexane/ethylacetate=3/1); ¹H NMR (500 MHz, CDCl₃) δ 3.88 (s, 3H), 4.16 (s, 2H), 4.39(s, 2H), 5.18 (dd, 1H, J=1.0, 10.5 Hz), 5.30 (dd, 1H, J=2.0, 10.5 Hz),5.67 (dd, 1H, J=1.0, 17.5 Hz), 6.25 (dd, 1H, J=2.0, 17.5 Hz), 6.47 (dd,1H, J=10.5, 17.5 Hz), 6.50 (dd, 1H, J=10.5, 17.5 Hz), 6.90 (dd, 1H,J=2.5, 8.0 Hz), 7.20-7.26 (m, 5H), 7.29-7.35 (m, 2H); ¹³C NMR (126 MHz,CDCl₃) δ 41.2, 55.6, 110.5, 113.8, 115.4, 119.3, 127.2, 128.7 (2C),129.2 (2C), 129.8, 132.1, 133.0, 135.1, 137.0, 138.3, 140.3, 141.9,144.8, 151.4, 159.8.

Synthesis Example 6N-(3-Benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazin-2-yl)acetamide(10)

Under an argon atmosphere, to a solution of2-amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine (9) (100mg, 292 mmol) in anhydrous pyridine (20 mL) were successively added4-(dimethylamino)pyridine (DMAP) (3.0 mg, 25 μmol) and acetyl chloride(65.0 μL, 911 μmol) at room temperature. The mixture was heated at 60°C. and stirred for an hour. After cooling to room temperature, to themixture was added water and the product was extracted with ethyl acetate(×3). The combined organic extract was washed successively with water(×1) and brine (×1), followed by drying over anhydrous sodium sulfate.After filtration and concentration under reduced pressure, the residuewas purified by silica gel flash column chromatography (n-hexane/ethylacetate=3/1) to give

N-(3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazin-2-yl)acetamide(10) (89.5 mg, 232 μmol, 79.7%) as a yellow solid. TLC R_(f)=0.17(n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz, CDCl₃) δ 2.33 (s, 3H),3.89 (s, 3H), 4.28 (s, 2H), 5.19 (dd, 1H, J=1.0, 10.8 Hz), 5.39 (dd, 1H,J=2.0, 10.8 Hz), 5.66 (dd, 1H, J=1.0, 17.2 Hz), 6.30 (dd, 1H, J=2.0,17.2 Hz), 6.45 (dd, 1H, J=10.8, 17.2 Hz), 6.52 (dd, 1H, J=10.8, 17.2Hz), 6.94 (dd, 1H, J=2.6, 8.4 Hz), 7.19-7.24 (m, 5H), 7.27-7.32 (m, 2H).

Synthesis Example 7N-(2-Benzyl-8-methoxybenzo[f]quinoxalin-3-yl)acetamide (11)

Under an argon atmosphere, to a solution of Hoveyda-Grubbs secondgeneration catalyst (61.3 mg, 97.8 μmol) in 1,2-dichloromethane (150 mL)was addedN-(3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazin-2-yl)acetamide(10) (348 mg, 903 μmol) at room temperature. The mixture was then heatedto 90° C. and stirred overnight (17 hours). After cooling to roomtemperature and concentration under reduced pressure, the residue waspurified by silica gel flash column chromatography (n-hexane/ethylacetate=4/3→1/1) to giveN-(2-benzyl-8-methoxybenzo[f]quinoxalin-3-yl)acetamide (11) (277 mg, 775mot, 85.8%) as a white solid. TLC R_(f)=0.27 (n-hexane/ethylacetate=1/1); ¹H NMR (400 MHz, CDCl₃) δ 2.33 (s, 3H), 4.00 (s, 3H), 4.50(s, 2H), 7.24-7.34 (m, 5H), 7.38 (dd, 1H, J=2.6, 8.8 Hz), 7.42 (br, 1H),7.90 (d, 1H, J=8.8 Hz), 7.93 (d, 1H, J=8.8 Hz), 9.01 (d, 1H, J=8.8 Hz).

Synthesis Example 8N-(2-Benzyl-8-hydroxybenzo[f]quinoxalin-3-yl)acetamide (13)

Under an argon atmosphere, to a suspension ofN-(2-benzyl-8-methoxybenzo[f]quinoxalin-3-yl)acetamide (60.0 mg, 168μmol) in anhydrous dichloromethane (2 mL) was added boron trifluoride(1.0 M in dichloromethane) (840 μL, 840 μmol) at 0° C. After elevatingto room temperature, the mixture was stirred for 3 hours. To the mixturewas then added a solution of saturated sodium hydrogencarbonate, and theproduct was extracted with ethyl acetate (×3). The combined organicextract was washed successively with water (×1) and brine (×1), followedby drying over anhydrous sodium sulfate. Filtration and concentrationunder reduced pressure gaveN-(2-benzyl-8-hydroxybenzo[f]quinoxalin-3-yl)acetamide (13) as a crudeyellow solid product. The product was used for the subsequent reactionwithout any further purification. TLC R_(f)=0.36 (n-hexane/ethylacetate=1/2); ¹H NMR (500 MHz, CD₃OD) δ 2.17 (s, 3H), 4.50 (s, 2H),7.20-7.32 (m, 9H), 7.78 (d, 1H, J=9.0 Hz), 7.96 (d, 1H, J=9.0 Hz), 9.44(d, 1H, J=7.5 Hz).

Synthesis Example 9 3-Amino-2-benzylbenzo[f]quinoxalin-8-ol (14,v-coelenteramine)

A solution of the obtained crude product ofN-(2-benzyl-8-hydroxybenzo[f]quinoxalin-3-yl)acetamide (13) in methanol(20 mL) was heated to 65° C. and stirred overnight (14 hours). Aftercooling to room temperature, the solution was concentrated under reducedpressure and the residue was purified by silica gel flash columnchromatography (n-hexane/ethyl acetate=1/1→1/10) to give3-amino-2-benzylbenzo[f]quinoxalin-8-ol (14, v-coelenteramine) (51.3 mg,170 mmol, quant., two steps) as an ocherous solid. TLC R_(f)=0.34(n-hexane/ethyl acetate=1/1); ¹H NMR (500 MHz, CD₃OD) δ 4.33 (s, 2H),7.16-7.20 (m, 2H), 7.21-7.26 (m, 1H), 7.30-7.38 (m, 5H), 7.50 (d, 1H,J=9.0 Hz), 7.76 (d, 1H, J=9.0 Hz), 8.77 (d, 1H, J=9.0 Hz).

Synthesis Example 10 3-Amino-2-benzylaline (12)

Under an argon atmosphere, to a solution of Hoveyda-Grubbs secondgeneration catalyst (84.5 mg, 135 μmol) in 1,2-dichloroethane (100 mL)was added 2-amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine(9) (585 mg, 1.70 mmol) at room temperature. The mixture was then heatedto 90° C. and stirred overnight (17 hours). After cooling to roomtemperature, the mixture was concentrated under reduced pressure. Theresidue was purified by silica gel flash column chromatography(n-hexane/ethyl acetate=3/1-2/1) to give3-amino-2-benzyl-8-methoxybenzo[f]quinoxaline (12) as a white solid (136mg, 431 μmol, 25.3%). TLC R_(f)=0.64 (n-hexane/ethyl acetate=1/1); ¹HNMR(500 MHz, CDCl₃) δ 3.98 (s, 3H), 4.39 (s, 2H), 4.63 (s, 2H), 7.21-7.38(m, 7H), 7.63 (d, 1H, J=9.0 Hz), 7.85 (d, 1H, J=9.0 Hz), 9.00 (d, 1H,J=9.0 Hz); ¹³C NMR (126 MHz, CDCl₃) δ 41.8, 55.4, 107.5, 118.1, 124.9,125.5, 125.8, 127.2, 128.7 (2C), 129.1 (2C), 130.1, 132.7, 134.6, 136.6,138.9, 143.1, 151.2, 158.5.

Synthesis Example 11 3-Amino-2-benzylbenzo[f]quinoxalin-8-ol (14,v-coelenteramine) (Synthesis from 12)

A mixture of 3-amino-2-benzyl-8-methoxybenzo[f]quinoxaline (12) (125 mg,396 μmol) and pyridinium hydrochloride (2.50 g, 21.6 mmol) was stirredat 190° C. (oil bath temperature) for 30 minutes and at 210° C. (oilbath temperature) for further 30 minutes. After cooling to roomtemperature, to the mixture was added water, the product was extractedwith ethyl acetate (×3). The combined organic extract was washedsuccessively with water (×1) and brine (×1), followed by drying overanhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by silica gel flash columnchromatography (n-hexane/ethyl acetate=1/1→1/10) to give3-amino-2-benzylbenzo[f]quinoxalin-8-ol (14, v-coelenteramine) (55.5 mg,184 μmol, 46.5%) as an ocherous solid.

Synthesis Example 12 v-Coelenterazine (2, v-CTZ)

Under an argon atmosphere, to a solution of3-(4-(tert-butyldimethylsilyloxy)phenyl)-1,1-diethoxypropan-2-one (15)(101 mg, 286 μmol) in ethanol (2 mL) and water (0.4 mL) was added3-amino-2-benzylbenzo[f]quinoxalin-8-ol (14) (55.5 mg, 184 μmol). Aftercooling to 0° C., to the mixture was further added conc. hydrochloricacid (0.20 mL). The mixture was heated to 80° C. and stirred overnight(18 hours). After cooling to room temperature and concentration underreduced pressure, the residue was purified by silica gel flash columnchromatography (n-hexane/ethyl acetate=1/1→1/2→ethyl acetate alone) inan argon flow to give v-coelenterazine (2, v-CTZ) (10.9 mg, 24.4 mol,13.2%) as ocherous powders. TLC R_(f)=0.50 (n-hexane/ethyl acetate=1/1);¹H NMR (500 MHz, CD₃OD) δ 4.08 (s, 2H), 4.51 (s, 2H), 6.69-6.72 (AA′BB′,2H), 7.17-7.24 (m, 5H), 7.29 (t, 2H, J=7.5 Hz), 7.43 (d, 2H, J=7.5 Hz),7.63 (d, 1H, J=8.5 Hz), 8.40 (br, 1H), 9.26 (br, 1H).

Synthesis Example 13 cf3-v-coelenterazine (17, cf3-v-CTZ)

Under an argon atmosphere, to a solution of3-(4-(trifluoromethyl)phenyl)propan-1,1-diethoxy-2-one (16) (86.9 mg,299 μmol) in ethanol (2 mL) and water (0.2 mL) was added3-amino-2-benzylbenzo[f]quinoxalin-8-ol (14) (60.2 mg, 200 μmol). Aftercooling to 0° C., to the mixture was further added conc. hydrochloricacid (0.10 mL). The mixture was heated to 80° C. and stirred overnight(13 hours). After cooling to room temperature and concentration underreduced pressure, the residue was purified by silica gel flash columnchromatography (n-hexane/ethyl acetate=1/1→1/2 ethyl acetate alone) inan argon flow to give cf3-v-coelenterazine (17, cf3-v-CTZ) (28.6 mg,57.3 μmol, 28.7%) as yellow powders. TLC R_(f)=0.24 (n-hexane/ethylacetate=1/2); ¹H NMR (500 MHz, CD₃OD) δ 4.26 (s, 2H), 4.51 (s, 2H),7.18-7.25 (m, 3H), 7.28-7.33 (m, 2H), 7.40-7.44 (AA′BB′, 2H), 7.54-7.66(m, 5H), 8.29 (br, 1H), 9.17 (br, 1H).

Example 1 Construction of Expression Vector

1) Construction of Renilla Luciferase Expression Vector pCold-RL

Novel expression vector pCold-RL was constructed by the followingprocedures. Using the vector pHis-RL having Renilla luciferase gene,described in Inouye & Shimomura, Biochem. Biophys. Res. Commun., 233(1997) 349-353, as a template, PCR was performed (cycle conditions: 25cycles; 1 min/94° C., 1 min/50° C., 1 min/72° C.) with a PCR kit(manufactured by Takara Bio Inc.) using two PCR primers, RL-17N/SacI (5′gcc GAG CTC ACT TCG AAA GTT TAT GAT CC 3′: SEQ ID NO: 21) andRL-18C/XhoI (5′ cgg CTC GAG TTA TTG TTC ATT TTT GAG AA 3′: SEQ ID NO:22). After purification by a PCR purification kit (manufactured byQiagen Inc.) and digestion with the restriction enzymes Sad and XhoI,the resulting fragment was inserted into the SacI/XhoI restrictionenzyme site of vector pCold-II (manufactured by Takara Bio Inc.,GenBank: AB186389) to give Renilla luciferase expression vectorpCold-RL. The nucleotide sequence of the insert DNA was confirmed by aDNA sequencer (manufactured by ABI Inc.).

2) Construction of Mutant Renilla Luciferase-547 Expression VectorpCold-hRL-547 to Shift the Emission to Longer Wavelength

The novel expression vector pCold-hRL-547 having mutant Renillaluciferase-547 gene (hRL-547) was constructed by the followingprocedures. The vector pCR2.1-hRL-547 plasmid having mutant Renillaluciferase-547 gene was constructed by the chemical synthesis and PCRprocedures. The mutant Renilla luciferase-547 gene was constructed asfollows: the construction of pCR2.1-hRL-547 involved designing based onRLuc8.6-547 described in Nature Methods, 4 (2007) 641-643, synthesizingby a combination of conventional chemical synthesis with PCR (OperonInc.) and cloning into the Asp718/EcoRI restriction enzyme site ofpCR2.1 (Invitrogen Inc.). In order to construct the pCold-hRL-547vector, pCR2.1-hRL-547 was digested with restriction enzymesAsp718/EcoRI and inserted into the Asp718/EcoRI restriction enzyme siteof vector pCold-II to construct Renilla luciferase-547 expression vectorpCold-hRL-547. The nucleotide sequence of the insert DNA was confirmedwith a DNA sequencer (manufactured by ABI Inc.).

The nucleotide sequence of DNA encoding hRL-547 is shown by SEQ ID NO:19. The amino acid sequence of hRL-547 is shown by SEQ ID NO: 20.

Example 2 Purification of Recombinant Protein

1) Expression of Recombinant Protein in Escherichia coli

To express the recombinant protein in Escherichia coli, Renillaluciferase gene expression vector pCold-RL and Renilla luciferase-547gene expression vector pCold-hRL-547 were used. The vectors weretransformed into Escherichia coli BL21 and the resulting transformantswere cultured in 10 mL of LB liquid medium (10 g of bactotryptone, 5 gof yeast extract and 5 g of sodium chloride, per liter of water, pH 7.2)supplemented with ampicillin (50 μg/m) and cultured at 37° C. for 16hours. The cultured LB broth was added to a fresh LB liquid medium of400 mL×5 (2 L in total) followed by incubation at 37° C. for 4.5 hours.After cooling on ice for an hour, 0.2 mM

IPTG was added and incubation was continued at 15° C. for 17 hours.After completion of the incubation, the cells were harvested bycentrifugation (5,000 rpm, 5 minutes) and used as the starting materialfor protein extraction.

2) Extraction of Recombinant Protein from Cultured Cells and NickelChelate Gel Column Chromatography

The culture cells collected were suspended in 200 mL of 50 mM Tris-HCl(pH7.6). Under cooling on ice, the cells were disrupted byultrasonication (manufactured by Branson, Sonifier Model Cycle 250) 3times each for 3 minutes. The cell lysate was centrifuged at 10,000 rpm(12,000×g) at 4° C. for 20 minutes. The soluble fractions obtained wereapplied onto a nickel-chelate column (Amersham Bioscience, column size:diameter 2.5×6.5 cm) to adsorb the recombinant protein. After washingwith 500 mL of 50 mM Tris-HCl (pH 7.6), the objective recombinantprotein was eluted with 50 mM Tris-HCl (pH 7.6) containing 0.1Mimidazole (manufactured by Wako Pure Chemical Industry). Proteinconcentration was determined using a commercially available kit(manufactured by Bio-Rad) by the method of Bradford and using bovineserum albumin (manufactured by Pierce) as a standard. The yields in thepurification process are shown in TABLE 1.

TABLE 1 shows the purification yields of Renilla luciferase and Renillaluciferase-547.

TABLE 1 Total Specific Total Total Activitiy Activity PurificationVolume Protein (×10⁸ rlu) (×10⁸ Process (mL) (mg) (%) (%) rlu/mg) 1)Renilla luciferase Soluble fraction 200 600 (100) 1423.0 (100)  2.37Dialysis after nickel 40 117.6 (19.6)  834.3 (58.6) 7.09 chelate gelelution 2) Renilla luciferase-547 Soluble fraction 200 480 (100)  79.9(100) 0.17 Dialysis after nickel 58.5 83.7 (17.4)  61.4 (76.9) 0.73chelate gel elution

Example 3 Measurement of Luminescence Activity

After Renilla luciferase (2.94 ng) or Renilla luciferase-547 (14.3 ng)was dispensed into a 96-well microplate (manufactured by Nunc) in anamount of 10 μL/well, 0.1 mL of 30 mM Tris-HCl (pH 7.6)-10 mM EDTAcontaining 0.05 μg of coelenterazine or v-coelenterazine was injectedinto the wells thereby to initiate the luminescence reaction, and theluminescence intensity was measured using a luminescence plate readerCentro LB960 (manufactured by Berthold). The luminescence intensity wasmeasured for 60 seconds 3 times in 0.1 second intervals, and the maximumluminescence intensity (I_(max)) and the mean (du) from luminescenceintegrated for 60 seconds were expressed in terms of relative activity(%) (TABLE 2).

TABLE 2 shows the luminescence activities of Renilla luciferase andRenilla luciferase-547.

TABLE 2 Maximum Luminescence Intensity I_(max) (Int.) (%) SubstrateRenilla Luciferase Renilla Luciferase-547 Coelenterazine 100 (100) 100(100) v-Coelenterazine 71.8 (47.3)  213 (73.4) cf3-v-Coelenterazine 18.9(12.3) 16.9 (11.9)

Example 4 Measurement of Emission Spectrum

The luminescence reaction was initiated by adding luciferase to 1 mL of50 mM Tris-HCl (pH 7.6)-10 mM EDTA containing 5 μg (1 μg/μL) ofsubstrate coelenterazine or v-coelenterazine dissolved in ethanol. Theprotein amounts of luciferases used were Renilla luciferase: 1.5 μg andRenilla luciferase-547: 7.2 μg in the case of coelenterazine assubstrate. On the other hand, the protein amounts in v-coelenterazine assubstrate were Renilla luciferase: 14.7 μg and Renilla luciferase-547:35.8 μg. Luminescence spectra were measured in a quartz cell with a 10mm optical path under the conditions of band width: 20 nm, sensitivity:medium, scan speed: 2000 nm/min and 22-25° C., using a fluorescencespectrophotometer FP-6500 (manufactured by JASCO) with the excitationlight source turned off. The emission maximum (λmax, m) and halfbandwidth (nm) were determined from the spectra measured, which areshown in TABLE 3.

TABLE 3 shows the results of analysis of the emission spectra of Renillaluciferase and Renilla luciferase-547.

TABLE 3 Emission Maximum λ_(max) (half bandwidth) (nm) Substrate Renillaluciferase Renilla luciferase-547 Coelenterazine 485.0 (95)  547.0 (124)v-Coelenterazine^(a) 519.0 (105) 593.0 (130) cf3-v-Coelenterazine 525.5(121) 599.0 (132)

As shown in EXAMPLES above, when v-coelenterazine was used as aluminescence substrate, the maximum emission wavelength was shiftedtoward a longer wavelength side relative to coelenterazine. In addition,when cf3-v-coelenterazine was used as a luminescence substrate, themaximum emission wavelength was shifted toward a longer wavelength sideeven relative to v-coelenterazine.

SEQUENCE LISTING FREE TEXT [SEQ ID NO: 1]

This is the nucleotide sequence of native apoaequorin.

[SEQ ID NO: 2]

This is the amino acid sequence of native apoaequorin.

[SEQ ID NO: 3]

This is the nucleotide sequence of native apoclytin-I.

[SEQ ID NO: 4]

This is the amino acid sequence of native apoclytin-I.

[SEQ ID NO: 5]

This is the nucleotide sequence of native apoclytin-II.

[SEQ ID NO: 6]

This is the amino acid sequence of native apoclytin-II.

[SEQ ID NO: 7]

This is the nucleotide sequence of native apomitrocomin.

[SEQ ID NO: 8]

This is the amino acid sequence of native apomitrocomin.

[SEQ ID NO: 9]

This is the nucleotide sequence of native apobelin.

[SEQ ID NO: 10]

This is the amino acid sequence of native apobelin.

[SEQ ID NO: 11]

This is the nucleotide sequence of native apobervoin.

[SEQ ID NO: 12]

This is the amino acid sequence of native apobervoin.

[SEQ ID NO: 13]

This is the nucleotide sequence of Renilla luciferase.

[SEQ ID NO: 14]

This is the amino acid sequence of Renilla luciferase.

[SEQ ID NO: 15]

This is the nucleotide sequence of Oplophorus luciferase.

[SEQ ID NO: 16]

This is the amino acid sequence of Oplophorus luciferase.

[SEQ ID NO: 17]

This is the nucleotide sequence of Gaussia luciferase.

[SEQ ID NO: 18]

This is the amino acid sequence of Gaussia luciferase.

[SEQ ID NO: 19]

This is the nucleotide sequence of Renilla luciferase mutant.

[SEQ ID NO: 20]

This is the amino acid sequence of Renilla luciferase mutant.

[SEQ ID NO: 21]

This is the nucleotide sequence of PCR primer RL-17N/SacI.

[SEQ ID NO: 22]

This is the nucleotide sequence of PCR primer RL-18C/XhoI

1. A process for producing a v-coelenteramine compound represented bygeneral formula (XIV):

(wherein R¹ is hydrogen, a halogen, a substituted or unsubstitutedhydrocarbon group, or a substituted or unsubstituted heterocyclicgroup), which comprises: (1) the step of reacting a compound representedby general formula (VIII):

(wherein R¹ is the same as defined above and R⁴ is a protecting group)with a methyltriphenylphosphonium salt in the presence of a base to givea compound represented by general formula (IX):

(wherein R¹ and R⁴ are the same as defined above), and, (2) the step ofperforming a ring-closing metathesis reaction on any one selected fromthe group consisting of the compound represented by general formula (IX)and a compound represented by general formula (X) which is the compoundrepresented by general formula (IX) wherein the amino is protected withR⁵:

(wherein R¹ and R⁴ are the same as defined above and R⁵ is a protectinggroup), and then deprotecting R⁴ and, if any, R⁵.
 2. A process forproducing a v-coelenterazine compound represented by general formula(II):

(wherein: R¹ is hydrogen, a halogen, a substituted or unsubstitutedhydrocarbon group, or a substituted or unsubstituted heterocyclic group,each of R² and R³ independently represents hydrogen, hydroxy, analkoxyl, a halogen, or a substituted or unsubstituted hydrocarbongroup), which comprises: (1) the step of reacting a compound representedby general formula (VIII):

(wherein R¹ is the same as defined above and R⁴ is a protecting group)with a methyltriphenylphosphonium salt in the presence of a base to givea compound represented by general formula (IX):

(wherein R¹ and R⁴ are the same as defined above), (2) the step ofperforming a ring-closing metathesis reaction on any one selected fromthe group consisting of the compound represented by general formula (IX)and a compound represented by general formula (X) which is the compoundrepresented by general formula (IX) wherein the amino is protected withR⁵:

(wherein R¹ and R⁴ are the same as defined above and R⁵ is a protectinggroup), and then deprotecting R⁴ and, if any, R⁵ to give av-coelenteramine compound represented by general formula (XIV):

(wherein R¹ is the same as defined above), and, (3) the step of reactingthe compound represented by general formula (XIV) with a compoundrepresented by general formula (XV):

(wherein each of R^(2′) and R^(3′) independently represents hydrogen,hydroxy, an alkoxyl, a halogen, a hydrocarbon group, or a hydroxy groupprotected with a protecting group) to give the compound represented bygeneral formula (II).
 3. The method according to claim 1, wherein atleast one base selected from the group consisting of n-butyl lithium,potassium tert-butoxide, sodium methoxide, sodium ethoxide and lithiumdiisopropylamide is used as the base in the step (1) above.
 4. Themethod according to claim 1, wherein at least one solvent selected fromthe group consisting of tetrahydrofuran, diethyl ether, cyclopropylmethyl ether, tert-butyl methyl ether, dioxane and toluene is used inthe step (1) above.
 5. The method according to claim 1, wherein thereaction temperature and reaction time in the step (1) above are set at0° C. to 40° C. for an hour to 4 hours.
 6. The method according to claim1, wherein a second generation Hoveyda-Grubbs catalyst is used as thecatalyst for the ring-closing metathesis reaction in the step (2) above.7. The method according to claim 1, wherein at least one solventselected from the group consisting of dichloroethane, dichloromethane,chloroform, trichloroethane, tetrachloroethane, benzene, toluene,xylene, monochlorobenzene, dichlorobenzene, hexane, heptane, octane,tetrahydrofuran, dioxane, diethyl ether, dibutyl ether, diisopropylether and dimethoxyethane.
 8. The method according to claim 1, whereinthe reaction temperature and reaction time of the ring-closingmetathesis reaction in the step (2) above are set at 25° C. to 110° C.for an hour to 48 hours.
 9. The method according to claim 1, wherein R¹is hydrogen, a halogen, a substituted or unsubstituted aryl, asubstituted or unsubstituted arylalkyl, a substituted or unsubstitutedarylalkenyl, an alkyl which may optionally be substituted with analicyclic group, an alkenyl which may optionally be substituted with analicyclic group, an alicyclic group, a heterocyclic group, or an alkynylwhich may optionally be substituted with an alicyclic group, in theformulae above.
 10. The method according to claim 1, wherein each of R²and R³ independently represents hydrogen, hydroxy, a halogen, an alkylhaving 1 to 4 carbon atoms which may optionally substituted with analicyclic group, trifluoromethyl or an alkoxyl, in the formula above.11. The method according to claim 1, wherein R⁴ is methyl,methoxymethyl, tetrahydropyranyl, benzyl, 4-methoxybenzyl,tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl,phenyldimethylsilyl, tert-butyldiphenylsilyl or triisopropylsilyl, inthe formulae above.
 12. The method according to claim 1, wherein R⁵ isacetyl, benzoyl, p-tosyl, tert-butoxycarbonyl or benzyloxycarbonyl, inthe formulae above.
 13. The method according to claim 2, wherein each ofthe protecting groups for the hydroxy groups for R^(2′) and R^(3′)independently represents tert-butyldimethylsilyl, methoxymethyl,tetrahydropyranyl, trimethylsilyl, phenyldimethylsilyl, triethylsilyl,phenyldimethylsilyl, tert-butyldiphenylsilyl or triisopropylsilyl, inthe formula above.
 14. The method according to claim 1, wherein thecompound represented by general formula (VIII) above is obtained by: (1)reacting 2-amino-3,5-diburomo-6-chloropyrazine with R¹MgX (wherein R¹ isthe same as defined above and X is a halogen) and ZnCl₂ in the presenceof a palladium catalyst to give a compound represented by generalformula (V):

(wherein R¹ is the same as defined above), (2) reacting the compoundrepresented by general formula (V) with a compound represented bygeneral formula (VI):

(wherein R⁴ is the same as defined above), in the presence of apalladium catalyst and a base to give a compound represented by generalformula (VII):

(wherein R¹ and R⁴ are the same as defined above), and, (3) reacting thecompound represented by general formula (VII) with tributyl(vinyl)tin inthe presence of a palladium catalyst.